Novel Peptide Involved in Energy Homeostasis

ABSTRACT

The expression of a mRNA encoding a putative 76 amino acid, secreted protein (“Enho1”) was found to negatively correlate with fasting triglyceride and cholesterol levels. A recombinant adenovirus was used to increase the expression of Enho1 mRNA in two mouse models of obesity, KK-A y  and Lep ob /Lep ob  mice. Over-expression of Enho1 by adenovirus injection significantly, and reproducibly, reduced fasting triglyceride and cholesterol levels in both models. In addition, transgenic mice strains were made that over express Enho1 protein. Additionally, the expression of a key gene involved in lipogenesis (fatty acid synthase) and FAS protein levels were reduced by ENHO1 adenoviral treatment in Lep ob /Lep ob  mice. Full-length ENHO1 peptide, or peptide derivatives, homologues, analogues, or mimetics thereof, delivered by oral intake, injection, subcutaneous patch, or intranasal routes, could be used as therapeutic or diagnostic agents for hypercholesterolemia, hypertriglyceridemia, insulin resistance, obesity, diabetes, and/or energy imbalance.

This application is a continuation of pending U.S. application Ser. No.12/848,308, now allowed, which is a continuation of U.S. applicationSer. No. 12/012,627, abandoned, which is a continuation-in-part ofexpired international (PCT) application No. PCT/US2006/030686, filedAug. 7, 2006, designating the United States, which application claimspriority to U.S. provisional application 60/705,940 filed Aug. 5, 2005.

TECHNICAL FIELD

This invention pertains to a novel gene and resulting protein, named“Energy Homeostasis Associated-1” (Enho1) which was found to be involvedin the association of obesity with insulin resistance and lipidemia.

BACKGROUND ART

Obesity is an increasingly prevalent global disease and has reachedepidemic proportions. Current estimates suggest that at least 50% of theWestern population is either overweight or obese. Obesity, particularlyabdominal obesity, combined with other conditions such as insulinresistance, dyslipidemia, hepatic steatosis, and hypertension is knownas the Metabolic, or Insulin Resistance, Syndrome. The centralpathophysiological features of the dyslipidemia associated with insulinresistance and type 2 diabetes are increased plasma triglycerides (TG)in very low density lipoproteins (VLDL), and reduced high densitylipoprotein (HDL) cholesterol. Commonly, increased circulating TGs arehydrolyzed into free fatty acids (FFA) and are taken up by peripheraltissues including the liver and can lead to hepatic steatosis, ornon-alcoholic fatty liver. Studies of several mutant mouse models ofobesity and metabolic disorders suggest that the link between insulinresistance and dysregulated TG is complex and involves both peripheraland central factors.

Insulin resistance refers to reduced insulin-stimulated glucose uptakein skeletal muscle and fat, and an impaired suppression of liver glucoseoutput (2). Hyperglycemia and hyperlipidemia are both side effects of,and causative agents in, the pathophysiology of type 2 diabetes.Glucotoxicity and lipotoxicity further promote insulin resistance andtype 2 diabetes due to suppression of insulin action and secretion fromthe 13-cell. Hyperinsulinemia is initially successful in suppressingliver glucose output, however the deleterious effects of increasedinsulin offset the gains associated with maintaining normal bloodglucose levels (2). Hyperinsulinemia is thought to be a factor in acluster of metabolic abnormalities, including hypertension,non-alcoholic fatty liver disease (NAFLD) and coronary heart disease(2). NAFLD disease is commonly associated with insulin resistance, andrequires two transcription factors: sterol regulatory element bindingprotein-1c (SREBP1c) and peroxisome proliferator receptor-γ (PPARγ)(3-6). Absence of SREBP1, or PPARγ signaling in liver inhibits thedevelopment of liver steatosis that occurs in obese insulin resistantmice (5-7).

Defining a common mechanism explaining insulin resistance has beendifficult because of the complexity of the insulin receptor (IR)signaling system, and the realization that it is not one, but manyfactors that contribute to the development of this disorder. Thetyrosine phosphorylation of two adaptor proteins, IRS 1 and IRS2, is acritical early step in the stimulation of glucose uptake by insulin(8-11). IRS1 and IRS2 have no intrinsic enyzmatic activity, and arethought to function as part of a molecular scaffold that facilitates theformation of complexes of proteins with kinase, phosphatase or ubiquitinligase function (12). Stimulation of phosphoinositide 3′ kinase (PI3K)by association with the IRS is a critical step in insulin-stimulatedglucose uptake. Activation of the p110 catalytic subunit of PI3Kactivates the lipid kinase domain, which phosphorylatesphosphatidylinositol-4,5-bisphosphate. Activation of PI3K is necessaryfor full stimulation of glucose uptake by insulin, although otherpathways might also be involved (12).

A metabolic state conducive to the development of insulin resistance isthought to result from an imbalance of caloric intake with oxidativemetabolism (13,14). Studies suggest that reduced mitochondrial functionin muscle is a factor in the development of insulin resistanceassociated with obesity (14,15). Stimulation of energy expenditure andsuppression of appetite both result in improved glucose metabolism inmouse models of obesity and type 2 diabetes. A well-characterizedexample of this is the adipocytokine leptin. Leptin acts in thehypothalamus and hindbrain to suppress appetite and through stimulationof the autonomic nervous system increases oxidative metabolism inskeletal muscle (16-20). However, leptin can also improve hepaticinsulin sensitivity independently of marked effects on food intake orbody weight (17).

Infusion of fatty acids (FA) is associated with rapid reductions ininsulin sensitivity in muscle within 4-6 h (21-23). The exact mechanismby which FA's reduce insulin-stimulated glucose uptake remains a matterof debate. Recent data indicate that FA's interfere with the IR signaltransduction pathways that stimulate glucose uptake (21,22,24). Onehypothesis is that an increase in the intracellular concentration ofFA's and diacyl-glycerol leads to the activation of a serine kinase,protein-kinase Cθ (PKCθ) (25). Phosphorylation of IRS1 on Ser³⁰⁷ by PKCθinhibits the phosphorylation of IRS-1 by the IR, leading to reducedactivation of PI3K and a reduction in the stimulation of glucose uptakeby insulin.

Abnormal Activity of Secreted Polypeptides as a Link Between Obesity andInsulin Resistance.

Determining mechanisms linking obesity with insulin resistance isimportant for developing new glucose lowering therapies. Recent researchinvestigating insulin resistance has focused on adipocytes. Obesity isassociated with aberrant regulation and function of a regulatory networkof polypeptides secreted from adipocytes (adipocytokines).Adipocytokines such as leptin, adiponectin, and resistin regulatehepatic glucose production, glucose disposal in muscle, and theproliferation and storage of lipid in adipocytes (26). Leptin regulatesenergy homeostasis through effects on neurons located in thehypothalamus and hindbrain, regulating ingestive behavior, autonomicnervous activity, and neuroendocrine system that govern metabolism(thyroid, adrenals) (16). Leptin resistance or reduced serum adiponectinassociated with obesity are factors that contribute to insulinresistance, through diminished insulin-sensitizing actions and byincreasing risk for developing steatosis (intracellular fatty acidaccumulation) (27,28). Non-adipose tissues also secrete peptides thataffect energy metabolism and insulin sensitivity, such as musclin frommuscle (29) and angiopoietin-related growth factor from liver (30).These factors may also be targets for the treatment of the metabolicsyndrome.

Melanocortin Receptor Knockouts for Investigating the Link BetweenObesity and Insulin Resistance:

Two melanocortin receptors expressed in areas of the central nervoussystem are involved in energy homeostasis. Targeted deletion of theneuronal melanocortin-4 receptor (MC4R) gene in mice (Mc4r−/− or Mc4rKOmice) causes obesity and hyperinsulinemia, and is also associated withincreased hepatic lipogenic gene expression and hepatic steatosis. Micedeficient for another neuronal melanocortin receptor (Mc3r−/− or Mc3rKOmice) develop a similar degree of obesity to Mc4r−/− mice when fed ahigh fat diet, but do not exhibit the same level of insulin resistance,hyperlipidemia and increased hepatic steatosisWork Mc3rKO and Mc4rKO onthe C57BL/6J (B6) strain both exhibit an exaggerated diet-inducedobesity, however the deterioration of insulin sensitivity in Mc4rKO ismore rapid and severe (31,32). FIGS. 1A-1E illustrate some of the knowndifferences in wild-type mice (C57BL/6J) and the two knockout mice interms of body mass as a function of either a low fat diet or a high fatdiet. (31,32) Severe insulin resistance in mice and humans is associatedwith hepatomegaly and steatosis, with increased hepatic lipogenesis(33). Mc4rKO develop hepatic insulin resistance and hepatomegaly in theobese state, and on a high fat diet (HFD) exhibit a marked deteriorationof glucose homeostasis associated with severe glucose and insulinintolerance. FIGS. 2A-2E show the differences in hepatomegaly andsteatosis in the two mouse strains, and also differences in expressionof genes involved in lipid metabolism. (4,17,57) On the other hand,Mc3rKO matched to Mc4rKO for fat mass (FM) exhibit a very modestimpairment of glucose homeostasis.

Sequences of cDNA Similar to ENHO1.

A sequence and putative open reading frame of a cDNA encoding a putativeprotein homologous to ENHO1 have previously been published by severalconsortiums involved in large-scale sequencing of cDNAs. See R. L.Stausberg et al., “Generation and initial analysis of more than 15,000full-length human and mouse cDNA sequences,” Proc. Natl. Acad. Sci.U.S.A., vol. 99, pp. 16899-16903 (2002) (Genbank accession number:BC021944, cDNA with complete coding sequence); and H. F. Clark et al.,“The secreted protein discovery initiative (SPDI), a large-scale effortto identify novel human secreted and transmembrane proteins: abioinformatics assessment,” Genome Res., vol. 13, pp. 2265-2270 (Genbankaccession number: NM_(—)198573, cDNA with complete coding sequence). Aprotein with similar homology for the first 37 amino acid residues ofSEQ ID NO:2 has been identified. However, the nucleotide and amino acidsequence of this described protein may be incorrect, due to a singlenucleotide error in the sequencing of the cDNA.

DISCLOSURE OF INVENTION

We have discovered a novel secreted peptide (Enho1) based on aninvestigation using two transgenic murine models of obesity. Usingmicroarray gene expression analysis and validation by RealTimequantitative PCR, the expression of an mRNA (genbank accession number:AK009710) encoding a putative 76 amino acid, secreted protein was foundto be reduced 10-fold in severely insulin resistant and glucoseintolerant Mc4rKO and Leptin-deficient (Lep^(ob)/Lep^(ob)) mice. Incontrast, in obese Mc3rKO mice, which are moderately glucose intolerantbut exhibit a normal response to insulin, there was a modest 30-40%reduction in the expression of the Enho1 protein. In C57BL/6J mice, anegative correlation was found in the hepatic expression Enho1 mRNA withfasting glucose levels. The expression of Enho1 in the hypothalamus alsodeclined with obesity and insulin resistance. We also confirmed that themRNA encoded a secreted protein. Based on the negative effect ofdiet-induced obesity and insulin resistance on Enho1 mRNA expression inliver and brain, the gene encoding the protein was initially designated“Swirl” (“suppressed with insulin resistance”), but was later renamed“Enho1” (“energy homeostasis associated-1”). A recombinant adenoviruswas used to increase the expression of ENHO1 in mouse models of obesity.Over-expression of ENHO1 by adenovirus injection significantly andreproducibly reduced fasting insulin, triglyceride and cholesterollevels. Additionally, the expression of a key gene involved inlipogenesis (fatty acid synthase) and FAS protein levels were reduced byENHO1 adenoviral treatment in Lep^(ob)/Lep^(ob) mice.

A transgenic FVB/NJ strain of mouse was created which over expresses theEnho1 open reading frame, using Enho1 DNA (SEQ ID NO:1) controlled bythe human β-actin promoter which is expressed in all tissues. FemaleFVB/NJ mice over expressing Enho1 had a significant reduction in fatmass, and a higher metabolic rate determined by measuring oxygenconsumption (VO2) using indirect calorimetry (Oxymax, ColumbusInstruments, Columbus, Ohio). The increase in metabolic rate observed inthe transgenic mice had been predicted, based on the results fromexperiments using recombinant adenovirus expressing Enho1. Mice infectedwith recombinant adenovirus expressing Enho1 lost more weight during anovernight fast, suggesting an impaired ability to reduce metabolic rateto compensate during fasting. FVB/NJ Enho1 transgenic mice exhibit thesame exaggerated weight loss during a fast, associated with a highermetabolic rate. A component of Enho1's anti-diabetic actions maytherefore involve stimulation of pathways involved in oxidativemetabolism. That is, Enho1 may improve the metabolic profile of obese,insulin resistant individuals partially through normalizing the balanceof kJ consumption with kJ expended through effects on physical activity,basal metabolic rate, or a combination of both.

Full-length Enho1 peptide, or peptide derivatives, homologues,analogues, or mimetics thereof, delivered by oral intake, injection,subcutaneous patch, or intranasal routes, could be used as therapeuticor diagnostic agents for hypercholesterolemia, hypertriglyceridemia,insulin resistance, obesity, diabetes, and/or disorders of energyimbalance.

Antibodies (AB1 and AB2) were raised against peptide fragments derivedfrom the open reading frame predicted for BC021944, and shown in FIG. 5as SEQ ID NO:8 and SEQ ID NO:9, respectively. These antibodies were usedto verify the presence of Enho1-immunoreactivity in human serum and inrat brain, strongly supporting the conclusion that the open readingframe predicted for BC021944 encodes a small secreted peptide.Enho1-immunoreactivity was detected in human plasma. (data not shown) Inrat brain, neurons with Enho1 immunoreactivity have been identified inthe arcuate nucleus of the hypothalamus. The significance of thisobservation is that neurons in the arcuate nucleus of the hypothalamushave been implicated in the regulation of energy expenditure, througheffects on both facultative thermogenesis and physical activity, and onglucose homeostasis [Cone R D. Anatomy and regulation of the centralmelanocortin system. Nat. Neurosci. 2005 May; 8(5):571-8; Coppari R,Ichinose M, Lee C E, Pullen A E, Kenny C D, McGovern R A, Tang V, Liu SM, Ludwig T, Chua S C Jr, Lowell B B, Elmquist J K. The hypothalamicarcuate nucleus: a key site for mediating leptin's effects on glucosehomeostasis and locomotor activity. Cell Metab. 2005 January;1(1):63-72.] The increased energy expenditure and physical activity ofthe FVB/NJ Enho1 transgenic mice may therefore involve action in thecentral nervous system, and more specifically through actions based onregulating activity of neurons in the arcuate nucleus of thehypothalamus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the differences in body mass in 6-month-old, femalemice after 12 weeks feeding either a low-fat diet (LF) or a high-fatdiet (HF) among three different strains, wild-type (WT), Mc3r −/−deficient mice (Mc3rKO), and Mc4r −/− deficient mice (Mc4rKO).(Significant effect of diet is indicated by “*” (p<0.001) or “#”(p<0.05); significant effects within diet indicated by letters, withgroups significantly different (p<0.05) given different letters;significance based on 2-way AVOVA)

FIG. 1B illustrates the differences in body weight gain as a percent ofstarting weight in 6-month-old, female mice after 12 weeks feedingeither a low-fat diet (LF) or a high-fat diet (HF) among three differentstrains, wild-type (WT), Mc3r −/− deficient mice (Mc3rKO), and Mc4r −/−deficient mice (Mc4rKO). (Significant effect of diet is indicated by “*”(p<0.001) or “#” (p<0.05); significant effects within diet indicated byletters, with groups significantly different (p<0.05) given differentletters; significance based on 2-way AVOVA)

FIG. 1C illustrates the differences in percent body fat in 6-month-old,female mice after 12 weeks feeding either a low-fat diet (LF) or ahigh-fat diet (HF) among three different strains, wild-type (WT), Mc3r−/− deficient mice (Mc3rKO), and Mc4r −/− deficient mice (Mc4rKO).(Significant effect of diet is indicated by “*” (p<0.001) or “#”(p<0.05); significant effects within diet indicated by letters, withgroups significantly different (p<0.05) given different letters;significance based on 2-way AVOVA)

FIG. 1D illustrates the differences in fat mass in 6-month-old, femalemice after 12 weeks feeding either a low-fat diet (LF) or a high-fatdiet (HF) among three different strains, wild-type (WT), Mc3r −/−deficient mice (Mc3rKO), and Mc4r −/− deficient mice (Mc4rKO).(Significant effect of diet is indicated by “*” (p<0.001) or “#”(p<0.05); significant effects within diet indicated by letters, withgroups significantly different (p<0.05) given different letters;significance based on 2-way AVOVA)

FIG. 1E illustrates the differences in fat-free mass in 6-month-old,female mice after 12 weeks feeding either a low-fat diet (LF) or ahigh-fat diet (HF) among three different strains, wild-type (WT), Mc3r−/− deficient mice (Mc3rKO), and Mc4r −/− deficient mice (Mc4rKO).(Significant effect of diet is indicated by “*” (p<0.001) or “#”(p<0.05); significant effects within diet indicated by letters, withgroups significantly different (p<0.05) given different letters;significance based on 2-way AVOVA)

FIG. 2A illustrates a comparison of liver histology, as shown in livercross-sections stained with hemotoxylin and eosin, from female mice feda high fat diet among three mice strains, wild-type (WT), Mc3r −/−deficient mice (Mc3rKO), and Mc4r −/− deficient mice (Mc4rKO).

FIG. 2B illustrates the differences in liver weight as a function ofadiposity (percent body fat) in mice fed both low and high fat dietsamong three mice strains, wild-type (WT), Mc3r −/− deficient mice(Mc3rKO), and Mc4r −/− deficient mice (Mc4rKO).

FIG. 2C illustrates the differences in mean liver weight (n=5-6/group)of male and female mice fed a moderate high fat diet among three micestrains, wild-type (WT), Mc3r −/− deficient mice (Mc3rKO), and Mc4r −/−deficient mice (Mc4rKO). (“*” indicates p<0.05 versus WT and Mc3rK0mice).

FIG. 2D illustrates the differences in expression of stearoyl-CoAdesaturase 1 (SCD1) in liver of mice fed a high fat diet among threemice strains, wild-type (WT), Mc3r −/− deficient mice (Mc3rKO), and Mc4r−/− deficient mice (Mc4rKO). Data is expressed as arbitrary units(a.u.). (“*” indicates p<0.05 versus WT and Mc3rK0 mice).

FIG. 2E illustrates the differences in expression of apolipoprotein A4(ApoA4) in liver of mice fed a high fat diet in three mice strains,wild-type (WT), Mc3r −/− deficient mice (Mc3rKO), and Mc4r −/− deficientmice (Mc4rKO). (“*” indicates p<0.05 versus WT and Mc3rK0 mice; “#”indicates p<0.05 versus WT mice, within gender).

FIG. 3A illustrates the amount of hepatic AK009710 mRNA expression(given as a percent of WT expression) in wild-type (WT) mice and inobese Mc3r −/− deficient mice (Mc3rKO).

FIG. 3B illustrates the amount of hepatic AK009710 mRNA expression(given as a percent of WT expression) in wild-type mice (WT), Mc4r −/−deficient mice (Mc4rKO), and leptin-deficient Lep^(ob)/Lep^(ob) mice(two models of obesity).

FIG. 3C illustrates the amount of hepatic AK009710 mRNA expression(given as a percent of expression with saline) in leptin-deficientLep^(ob)/Lep^(ob) mice injected 4 times over 2 days with either salineor leptin (0.5 mg/g).

FIG. 4 illustrates the nucleic acid sequence of the Enho1 gene (MouseAK009710; SEQ ID NO:1), with the open reading frame encoding the Enho1protein underlined, and its putative amino acid translation productEnho1 (SEQ ID NO:2).

FIG. 5 illustrates the alignment of the putative Enho1 protein sequencesfor mouse (SEQ ID NO:2), human (SEQ ID NO:14), rat (SEQ ID NO:12), dog(SEQ ID NO:15), pig (SEQ ID NO:16), cow(SEQ ID NO:17), sheep (SEQ IDNO:18), and chimpanzee (SEQ ID NO:13), showing the regions of theputative secreted polypeptide used to generate polyclonal antibodies(pAB1 ((SEQ ID NO:8) and pAB2(SEQ ID NO:9)).

FIG. 6A illustrates the results of a Northern blot analysis using aradioactive-labeled portion of the AK009710 DNA sequence encoding Enho1protein on human tissue samples (Lanes 1-8 represents RNA from heart,brain, placenta, lung, liver, skeletal muscle, kidney and pancreas,respectively).

FIG. 6B illustrates the relative intensity of Enho1 mRNA bands from aNorthern blot analysis using the full-length mouse AK009710 DNA probe onmouse tissue samples.

FIG. 6C illustrates the results of a Western blot analysis for FLAGimmunoreactivity in media (M) and cell lysates (Pel) from HEK293human-kidney derived cells transfected with pCMV-Enho1:Flag, pCMV-GFP,or in media from HEK293 cells infected with adenoviral vector expressingEnho1:Flag fusion protein [no transfected DNA (lanes 1,2); transfectedwith a pCMV-Enho1:FLAG construct (lanes 3,4); transfected withpCMV-Enho1 (lanes 5,6); or transfected with pCMV-GFP (lanes 7,8);infected with AdSEnho1:FLAG (lane 9); infected with AdSEnho1 (lane 10);or infected with Ad5GFP (lane 11)]. To visualize Enho1 protein, a fusionprotein was created with a C-terminal FLAG-epitiope tag.

FIG. 6D illustrates the results of a Western blot analysis for ENHO1protein in various tissues (liver, muscle, and brain) from Mc4r−/− miceinjected with Ad5-ENHO1:FLAG 4 days prior to the assay, along withcontrols, both positive (HEK293 cells infected with Ad5-ENHO1:FLAG) andnegative (Liver from non injected Mc4r−/− mice).

FIG. 7A illustrates the results of a Western blot analysis using lysatefrom HEK293 cells infected with recombinant AdSEnho1 (native protein) orAdSEnho1:FLAG (C-terminal FLAG-tagged fusion protein) after incubationwith a polyclonal antibody (pAB1) against the N-terminus of Enho1 (asshown in FIG. 5).

FIG. 7B illustrates the results of a Western blot analysis using lysatefrom HEK293 cells infected with recombinant AdSEnho1 (native protein) orAdSEnho1:FLAG (C-terminal FLAG-tagged fusion protein) after incubationwith a polyclonal antibody (pAB2) against the C-terminus of Enho1 (asshown in FIG. 5).

FIG. 7C illustrates the treatment protocol used to administer AdSEnho1or Ad5-GFP into the tail vein of various strains of mice to test theeffects of Enho1 treatment on mice metabolism (results presented inTable 1).

FIG. 8A illustrates the expression level of fatty acid synthase (Fasn)mRNA in liver from obese leptin-deficient (Lep^(ob)/Lep^(ob)) mice eightdays after injection with either Ad5-ENHO1 or Ad5-GFP. Data expressed inarbitrary units (AU) (n=6-8/group; “*” p<0.05).

FIG. 8B illustrates the expression level of stearoyl-CoA desaturase(Scd1) protein in liver from obese leptin-deficient (Lep^(ob)/Lep^(ob))mice eight days after injection with either Ad5-ENHO1 or Ad5-GFP. Dataexpressed in arbitrary units (AU) (n=6-8/group; “*” p<0.05).

FIG. 8C illustrates the expression level of acetyl CoA carboxylase (Acc)mRNA in liver from obese leptin-deficient (Lep^(ob)/Lep^(ob)) mice eightdays after injection with either Ad5-ENHO1 or Ad5-GFP. Data expressed inarbitrary units (AU) (n=6-8/group; “*” p<0.05).

FIG. 8D illustrates the expression level of a gene involved in insulinresistance (a suppressor of cytokine signaling) in liver from obeseleptin-deficient (Lep^(ob)/Lep^(ob)) mice eight days after injectionwith either Ad5-ENHO1 or Ad5-GFP. Data expressed in arbitrary units (AU)(n=6-8/group; “*” p<0.05).

FIG. 9A illustrates the construction of the transgene (BAP-Enho1) usedto generate transgenic mouse strains that over express Enho1.

FIG. 9B illustrates the amount of Enho1 expression in livers fromtransgenic mice pups (produced using the transgene of FIG. 9A) fromFVB/NJ founders at 5 weeks.

FIG. 9C illustrates the level of fasting triglycerides (TG) from serumof transgenic mice pups (produced using the transgene of FIG. 9A) fromFVB/NJ founders at 9 weeks.

FIG. 9D illustrates the body composition as measured by percent body fat(% body fat; left graph) and fat mass (showing both free fat mass (FFM)and fat mass (FM); right graph) in transgenic mice pups (produced usingthe transgene of FIG. 9A) from FVB/NJ founders at 5 and 9 weeks ascompared with control.

FIG. 9E illustrates change in fat mass (showing both free fat mass (FFM)and fat mass (FM)) of transgenic mice pups (produced using the transgeneof FIG. 9A) from FVB/NJ founders after 7 days on a 60% high fat diet.

FIG. 10 illustrates the results from a PCR screen for integration ofBAP-Enho1 into the genome of C57BL/6J pups (from injection of BAP-Enho1into C57BL/6J oocytes) showing six pups out of 23 with integration ofthe transgene into the genome.

FIG. 11A illustrates the increase in Erk phosphorylation in 3T3-L1adipocytes 15 minutes after the application of the secreted portion ofprotein Enho1 (Enho1³⁴⁻⁷⁶; SEQ ID NO:10).

FIG. 11B illustrates the increase in Erk phosphorylation in HepG2hepatocytes 15 minutes after the application of the secreted proteinEnho1 (Enho1³⁴⁻⁷⁶; SEQ ID NO:10).

FIG. 12A illustrates the association between hypothalamic Enho1 mRNAexpression and body weight in six-month-old mice from three strains(C57BL/6J(WT), Mc3rKO (Mc3r), or Mc4rKO (Mc4r) fed for three months onone of two diets (LF=10% kJ/fat; HF=60% kJ/fat).

FIG. 12B illustrates the association between hypothalamic Enho1 mRNAexpression and HOMA-IR [(fasting insulin×glucose)/22.5] in six-month-oldmice from three strains (C57BL/6J(WT), Mc3rKO (Mc3r), or Mc4rKO (Mc4r)fed for three months on one of two diets (LF=10% kJ/fat; HF=60% kJ/fat).

FIG. 12C illustrates the association between HOMA-IR [(fastinginsulin×glucose)/22.5] and hypothalamic Socs3 (suppressor of cytokinesignaling 3) mRNA expression in six-month-old mice from three strains(C57BL/6J(WT), Mc3rKO (Mc3r), or Mc4rKO (Mc4r) fed for three months onone of two diets (LF=10% kJ/fat; HF=60% kJ/fat).

FIG. 13A illustrates the physical activity (measured in beam breaks/10min) in wild-type (WT) and BAP-Enho1 transgenic mice (Tg) over 3 days.

FIG. 13B illustrates the physical activity (measured in average numberbeam breaks/10 min for the period) in dark and light periods inwild-type (WT) and BAP-Enho1 transgenic mice (Tg) over a 24 hr.

FIG. 13C illustrates whole body fat oxidation (RER) in dark and lightperiods in wild-type (WT) and BAP-Enho1 transgenic mice (Tg) over a 24hr.

FIG. 13D illustrates the metabolic rate (measured as VO2 (ml/h×10³)) indark and light periods in wild-type (WT) and BAP-Enho1 transgenic mice(Tg) over a 24 hr.

FIG. 13E illustrates the metabolic rate as a function of free fat mass(measured as VO2 (ml/h/gFFM×10³)) in dark and light periods in wild-type(WT) and BAP-Enho1 transgenic mice (Tg) over a 24 hr.

FIG. 13F illustrates the metabolic rate as a function of body weight(measured as VO2 (ml/h/gBW×10³)) in dark and light periods in wild-type(WT) and BAP-Enho1 transgenic mice (Tg) over a 24 hr.

MODES FOR CARRYING OUT THE INVENTION

The present invention discloses a novel secreted protein (Enho1) andidentifies some of its functions. The sequence of this protein was foundto be highly conserved across several mammalian species, and thesequences are shown in SEQ ID NOS:2 and 12-18. In addition the nucleicacids that encode this protein was used to make mice that over expressedthe Enho1 protein, either by infection with a recombinant adenovirusexpressing Enho1 or by making a transgenic strain using Enho1 DNAcontrolled by an actin promoter.

In one embodiment, the Enho1 protein, fragment, derivative or analogsare used therapeutically to prevent a pathophysiology associated withincreased body mass, e.g., obesity, hyperglycemia, hyperinsulinemia,insulin resistance, hyperlipidemia, and non-insulin dependent (type 2)diabetes mellitus.

In another embodiment, the purified Enho1 protein, fragment, derivativeor analog is isolated from various mammals, or made synthetically, ormade using cell cultures that express the protein.

In another embodiment, antibodies are made to the Enho1 protein, itsfragments, derivatives or analogs. These antibodies can be used in a kitto identify the Enho1 protein from various samples, including bodyfluids.

In another embodiment, transgenic animals are made that over express theEnho1 protein by linking the Enho1 sequence to an active promoter, e.g.,an actin promoter. In another embodiment, a transformation vectorcomprising at least the open reading frame of SEQ ID NO:1 (the portionunderlined in FIG. 4) are made.

Example 1 Discovery of Enho1 Protein and Its Function

Microarrays analyzing hepatic gene expression in lean and obese Mc3rKOwere performed using arrays printed from libraries of 16,463-18,40070-mer oligonucleotides (Mouse Array-Ready Oligo Set Version 2.0, QiagenOperon, Alameda, Calif.) (34-36). The microarray data indicatedincreased expression of genes involved in lipid metabolism(apoliproteins) and oxidative stress secondary to obesity.Interestingly, the expression of only three genes was reduced in liverof Mc3rKO irrespective of age, gender, and adiposity. Two of the genesencoded proteins with known function: neuraminidase 3 (neu3), encodingan enzyme that cleaves sialic acid from glycoproteins and glycolipids,and solute carrier family 21 (slc21a1) which encodes an organic aniontransporter. The third gene (AK009710) was novel and had no assignedfunction in the databases.

Quantitative RealTime PCR confirmed the microarray data showing atendency for a modest reduction in the expression of AK009710 in liverof Mc3rKO (FIG. 3A). Intriguingly, a more dramatic reduction in theexpression of AK009710 was observed in liver of severely insulinresistant Mc4rKO and leptin-deficient (Lep^(ob)/Lep^(ob)) mice (FIG.3B). In addition, short term treatment with leptin (4 injections of 0.5mg/g leptin over 2 days) significantly increased Enho1 in the liver ofLep^(ob)/Lep^(ob) mice. (FIG. 3C). Expression of AK009710 in 6 wk oldlean Mc4rKO is normal (data not shown), indicating that the decline inliver expression of Mc4rKO is secondary to the age-related onset ofobesity, insulin resistance and hepatic steatosis (32). Moreover,AK009710 expression correlated negatively with fasting glucose indiet-induced obese B6 mice (n=13, R²=0.67, P<0.001) (data not shown).

Overall, these results indicate the identification of a small secretedprotein whose expression in liver declines with insulin sensitivity andhepatic steatosis. Based on the observation that the decline in AK009710expression in liver correlates with the severity of hepatic steatosisand insulin resistance, and that this decline is reversed by an insulinsensitizor, the name “suppressed with insulin resistance 1” (swir1) waschosen, but later renamed “energy homeostasis associated-1” (Enho1).

Oligonucleotide primers targeted to AK009710 mRNA were utilized tomeasure AK009710 gene expression in liver cDNA from various mouse modelsof obesity and insulin resistance. Sequences of primers were: sense5-cctgagggtgctgtctgtcatg-3′ (SEQ ID NO:3), antisense5′-cagtagcagcaagaagcctacg-3′ (SEQ ID NO:4), probe5′-6FAM-ctctcatcgccatcgtctgca-BHQ-3′ (SEQ ID NO:5). In agreement withthe initial microarray result, AK009710 mRNA was down regulated in theliver of Mc3r−/− mice compared to WT mice (by 55%, p<0.05; FIG. 3A).Next was examined the AK009710 liver mRNA expression in two other modelsof obesity: Mc4r−/− mice and leptin-deficient Lep^(ob)/Lep^(ob) mice.AK009710 mRNA was expressed at approximately 10-fold lower levels inboth of these models compared to wild-type mice (WT) (FIG. 3B). When allanimals were grouped together negative relationships between AK009710mRNA expression and glucose and insulin values were observed (Data notshown). AK009710 gene expression was not altered in the liver of youngand aged WT mice fed ad libitum, fasted for 16 h, or fasted for 24 h andre-fed for 4 h. (data not shown) AK009710 is not thought to be alteredby nutritional status.

In order to determine whether AK009710 mRNA might be regulated secondaryto the development of insulin resistance, AK009710 mRNA was measured inthe liver of young, normoinsulinemic and older hyperinsulinemic Mc4r−/−mice. AK009710 mRNA was observed to be similar to the wild-type mice inyoung Mc4r−/− mice (average age ˜7 weeks), whereas it was significantlyreduced in older (−16 weeks) Mc4r−/− mice. (data not shown).Furthermore, AK009710 mRNA was also measured in a small number (n=4) ofhuman-derived hepatocytes from diabetic and non-diabetic subjects.Although the expression tended to be lower in the diabetic samples, thedifference was not statistically significant (data not shown).

Thus, while obesity of Mc3r−/− and Mc4r−/− mice is similar, insulinresistance and elevated serum lipids (triglyceride, cholesterol) areknown to be far more severe for Mc4r−/− mice. The reduction in AK009710gene expression in the liver correlated with the known severity ofinsulin resistance and hyperlipidemia in Mc4r−/− mice.

Example 2 Sequence and Bioinformatics Analysis

AK009710 is a mouse tongue cDNA clone 1247 by in length and belongs tothe Unigene cluster Mm.34074, 2310040A07Rik: RIKEN cDNA 2310040A07 gene.It hypothetically could encode a 534 amino acid protein immediately fromthe 5′ end of the sequence. Given that this putative protein did notstart with a methionine residue, it was listed as a truncated product.BLAST analysis of AK009710 for homologous EST or cDNA sequences on theNCBI database revealed a significant match with NM198573, a humantranscript discovered by a large-scale Secreted Protein DiscoveryInitiative encoding a hypothetical protein of 87 aa (UNQ470/GAAI470)(37). The match with human sequence —NM_(—)198573 was 85% identityacross 392 nucleotides, p=5e⁻⁸³. The human sequence encoded an 87 aminoacid protein in one exon. Translation of AK009710 in six frames revealedan open reading frame encoding a 76 amino acid protein (SEQ ID NO:2). Analignment between this putative mouse protein and the human proteinencoded by NM_(—)198573 (GAAI470, or UNQ470) revealed strong homologyover the first 37 residues. UNQ470/GAAI470 maps to chromosome 9p13.2.and is adjacent to the ciliary neurotrophic factor receptor (CNTFR)gene.

The sequence of AK009710 was verified using cDNA isolated from mouseliver. Moreover, an 868 by cDNA cloned from mouse liver by the NationalInstitutes of Health Mammalian Gene Collection (MGC) Program, with theaccession number BC021944, recently posted on the NCBI database agreeswith our sequencing data. (FIG. 4, SEQ ID NO:1) BLAST analysis revealedthat the predicted 76 aa sequence for mouse (SEQ ID NO:2) is highlyconserved in several mammalian species (FIG. 5; SEQ ID NO:12 (rat), SEQID NO:13 (chimpanzee); SEQ ID NO:14 (human); SEQ ID NO:15 (dog); SEQ IDNO:16 (pig); SEQ ID NO:17 (cow); SEQ ID NO:18 (sheep)). In FIG. 5, pAB1(SEQ ID NO: 8) and pAB2 (SEQ ID NO:9) refer to the regions of theputative secreted polypeptide used to generate polyclonal antibodies.The predicted signal sequence is underlined. In addition peptides weresynthesized corresponding to amino acids 34 through 76 (Enho1³⁴⁻⁷⁶; SEQID NO:10), and to amino acids 39 through 76 (Enho1³⁹⁻⁷⁶; SEQ ID NO:11)of SEQ ID NO:2, the mouse protein.

Further analysis of the putative mouse sequence from AK009710 and thehuman GAAI470 sequence revealed a single nucleotide gap in the humansequence on GenBank. Therefore a mouse AK009710 PCR product was clonedand sequenced in both directions to confirm which sequence, either themouse or the human, was correct. The sequence-verified AK009710 mouseclone revealed a 76 amino acid translation product (SEQ ID NO:2) (FIG.4). A publicly available Origene clone of human GAAI470 was analyzed andshown not to contain the nucleotide gap, indicating a sequencing errorin the NM_(—)198573 sequence on GenBank. Alignment of AK009710 sequencefrom different species, including human, revealed a 100% homologybetween rat, human, mouse and chimp sequences at the protein level (FIG.5).

Bioinformatics analysis revealed that this protein is highly likely tobe a secreted peptide (87% probability, SignalP 3.0) with the mostlikely cleavage site between positions 33 and 34. The human sequencecontains 1 possible serine site, and 1 possible threoninephosphorylation sites, all 3′ to the predicted cleavage point, as wellas 6 possible glycosylation sites, also all 3′ to the cleavage site.

Example 3 Tissue Distribution of Enho1

In order to examine the tissue distribution of AK009710 mRNA a Northernblot was performed using the 210 nt sequence containing the 76 aminoacid protein ENHO1 and a human Multiple Tissue Northern blot (BDBiosciences, Palo Alto, Calif.). As can be seen in FIG. 6A, a singleband of ˜1.35 kb was detected in humans in brain and liver samples only,with highest levels detected in the liver. However, as seen in FIG. 6B,Enho1 was found in mice in the liver, brain, and muscle.

Example 4 Confirmation that Enho1 is a Secreted Polypeptide

Bioinformatics analysis predicted the presence of a putative signalsequence suggesting that AK009710 is a secreted peptide. To test this,the coding sequence of the 76 amino acid protein was amplified frommouse liver cDNA by PCR using primers with Not1(5′-ggggcggccgcaccatgggggcagccatctcccaa-3′ (SEQ ID NO:6)) and Xho1(5′-gggctcgagggccagagcccttcagggctgcag-3′ (SEQ ID NO:7)) restrictionenzyme sites attached. The product was ligated into a pCMV-Tag1 vectorwith a FLAG epitope at the C-terminal end (pCMV-Enho1:FLAG), thentransiently transfected into HEK human kidney-derived cells. Thisproduct was also used to create two adenoviral constructs—one with Enho1attached to a FLAG epitope (Ad-Enho1:FLAG), and another without the FLAGepitope (Ad-Enho1).

HEK293 cells were transfected with pCMV-Enho1, or pCMVEnho1:FLAG. Mediawas collected after 16 h, immunoprecipitated, and then run on a 20%polyacrylamide gel to be visualized by an anti-FLAG antibody. Theseexperiments were repeated using recombinant adenovirus (Ad5) expressingnative or FLAG-tagged Enho1.

When HEK293 cells were untransfected (lanes 1, 2), or transfected withempty vector (lanes 5, 6), or vector containing GFP (lanes 7, 8) noFLAG-immunoreactive bands were visualized (FIG. 6C). However, when thepCMV-Enho1:FLAG construct was transfected into HEK293 cells (lanes 3,4), FLAG-positive immunoreactivity was detected in both the media andthe cell pellet, indicating that at least some of the transfected Enho1product was being actively secreted by the cells into the media. Similarresults were observed in the media of HEK293 cells infected withAd-Enho1:FLAG (FIG. 8, lane 9). Lanes 10 and 11 show no FLAGimmunoreactivity in media of cells infected with adenovirus containingEnho1 without the FLAG epitope, or with GFP, respectively. Thus thepresence of FLAG immunoreactivity in cultured media of HEK-293 cellstransfected with an expression vector expressing an epitope-taggedfusion protein (pCMV-Enho1FLAG) confirmed that an undefined portion ofthe 76 aa protein is secreted.

Most adenovirus injected in the tail vein infects liver (38). To testwhether tail vein injection of adenovirus leads to increased expressionof ENHO1 mRNA in the liver, as well as other tissues, three Mc4r−/− micewere injected with Ad5-ENHO1:FLAG adenovirus and the tissues collectedafter four days. Analysis by Western blot revealed the presence of FLAGimmunoreactivity in the liver, muscle and brain tissue indicating thepresence of ENHO1 in these tissues. (FIG. 6D) The distribution of Enho1FLAG immunoreactivity in tissues of mice infected with Ad5-Enho1FLAG isconsistent with a polypeptide secreted into the circulatory system fromliver. Both of these tests indicated that Enho1 is a secreted protein.

Example 5 Treatment of Obese Insulin Resistant Mice with Ad5-Enho1:Mc4rKO

Recombinant adenoviral vector-mediated expression has been used toinvestigate regulation of liver metabolism and insulin sensitivity(39-41). Three recombinant adenovirus vectors were constructedexpressing the 76 aa protein (Ad5-Enho1), a C-terminal FLAG-taggedfusion protein (Ad5-Enho1:FLAG), or green fluorescent protein for thenegative control (Ad5-GFP). Expression of protein following tail veininjection was confirmed using anti-FLAG antibody (FIG. 6D and data notshown). Synthesis of Enho1 was confirmed in HEK293 cells transfectedwith Ad5-Enho1 using polyclonal antibodies (pAB1 & pAB2 in FIG. 5)against N- and C-terminal regions of the putative secreted protein(Sigma Genosys, The Woodlands, Tex.) (FIG. 7A, B).

To investigate whether ENHO1 is involved in glucose metabolism,Ad5-ENHO1 or Ad5-GFP (5×10⁸ pfu) was injected into the tail vein of20-week-old male and female Mc4r−/− (n=3 of each sex per group). Bothgroups of animals were matched for body weight, glucose levels andglucose tolerance before Ad5-ENHO1 or Ad5-GFP injections. Anintraperitoneal glucose tolerance test (IPGTT) was performed one weekprior to injection of Ad5-ENHO1 or Ad5-GFP, and all mice were observedto be glucose intolerant. IPGTT were performed on mice after anovernight fast, with a single intraperitoneal injection of 1 g/kgglucose, and blood glucose measured with a blood glucose meter and teststrips (Glucometer Elite, Bayer Corp., Elkhart, Ind.) from the tailblood of the animals at several intervals, as described by W. Fan etal., “The Central Melanocortin System Can Directly Regulate SerumInsulin Levels,” Endocrinology, vol. 141, pp. 9 3072-3079 (2000).

Mice were injected with 5×10⁸pfu of Ad5-ENHO1 or Ad5-GFP in 100 μA ofdiluent (DMEM) into the tail vein. Animals were observed and weigheddaily throughout the 4-day experiment. On day 4 animals were givenanother IPGTT (0.4 mg glucose/g body weight).

Four days after injection of Ad5-ENHO1, the mice demonstrated improvedglucose tolerance as measured using IPGTT (data not shown). There was nochange in body weight, blood glucose or cholesterol levels throughoutthe experiment. There was a trend for a decrease in insulin and serumtriglyceride levels (Table 1).

TABLE 1 Characteristics of Mc4r−/− mice 4 days post adenoviral infusion.Body Choles- Triglyc- Weight Glucose Insulin terol erides Group (g)(mg/dL) (ng/mL) (mg/dL) (ng/mL) Ad5-ENHO1 50.4 ± 1.8 184 ± 10  5.9 ± 1.591 ± 7 26 ± 4 Ad5-GFP 49.5 ± 2.2 169 ± 16 10.4 ± 2.0 82 ± 6 38 ± 6

Example 6

Treatment of Obese Insulin Resistant Mice with Ad5-Enho1: B6 Ay/a

A second adenoviral experiment was performed whereby Ad5-ENHO1 orAd5-GFP was injected into the tail vein of male C57BL6 Ay/a mice (n=9per group) that had been maintained on a very high fat diet for ˜3months. Mice were given 1 week to recover from the injections, and thenan IPGTT was performed. Then the mice were given another week torecover, and an insulin tolerance test (ITT) was performed.

No differences in body weight (40.3±1.3 g for the Ad5-ENHO1 mice;41.4±1.2 g for the Ad5-GFP mice), fasting insulin (0.97±0.2 ng/mL forthe Ad5-ENHO1 mice; 1.13±0.2 ng/mL for the Ad5-GFP mice), or fastingglucose levels (126±7 mg/dL for the Ad5-ENHO1 mice; 139±8 mg/dL for theAd5-GFP mice) were observed at 20 days after injection. IPGTT 7 daysafter injection revealed a very modest improvement in glucose tolerancein the Ad5-ENHO1 group at the 30-minute (p=0.06) and 45-minute (p=0.15)time points. Changes in blood glucose levels 14 days after injectionwere significantly different between groups, being significantly reducedin the Ad5-ENHO1 group (data not shown). An ITT 14 days post-infectionrevealed no significant difference in insulin sensitivity at any timepoint; however Ki (calculated as glucose at 30 minutes post insulininjection subtracted from baseline glucose, divided by 30) wassignificantly different.

The lower fasting blood glucose levels observed in these mice 14 daysafter infection was not apparent at 21 days after infection. Inagreement with the first adenoviral experiment in Mc4r−/− mice, therewas a trend for a decrease in serum triglycerides in the Ad5-ENHO1 group(Table 2).

TABLE 2 Infection with Ad5-Enho1 is associated with evidence of improvedmetabolic profile in mouse models of obesity and insulin resistance.Liver Liver wgt lipid Total Serum Serum BW pre- BW post- Liver wgt as a% content liver lipid TG TC HOMA- Strain Treatment injection injection(g) BW (mg/g) (mg) (mg/dL) (mg/dL) IR OBOB GFP 64.0 ± 1.9 64.8 ± 2.0 4.7± 0.2 7.4 ± 0.3 142 ± 8  654 ± 25 63 ± 6 173 ± 6  308 ± 114 Enho1 65.0 ±2.2 64.6 ± 2.0 4.3 ± 0.2 6.7 ± 0.3 128 ± 7  552 ± 50  38 ± 4* 146 ± 8*157 ± 18  KKAy GFP 48.0 ± 1.9 46.7 ± 1.7 2.1 ± 0.2 4.7 ± 0.3 57 ± 10 117± 24 435 ± 53 214 ± 13 98 ± 19 Enho1 46.6 ± 1.2 45.3 ± 0.8 1.8 ± 0.1 4.3± 0.1 33 ± 2*  58 ± 5*  315 ± 16* 172 ± 9  62 ± 11 Ay/a GFP 45.2 ± 2.544.6 ± 2.1 2.2 ± 0.2 5.1 ± 0.2 119 ± 11  282 ± 39  71 ± 18 79 ± 3 40 ±6  Enho1 44.8 ± 1.4 44.5 ± 1.1 1.8 ± 0.2  4.2 ± 0.3* 81 ± 20  145 ± 41*53 ± 4 72 ± 2 28 ± 4  *P < 0.05 compared to Ad5-GFP treated controls(within strain), n = 5-8/group.

Example 7 Treatment of Obese Insulin Resistant Mice with Ad5-Enho1:Agouti (KK-A^(y)) Mice (Genetically Obese and Hyperlipidemic Mice)

To more specifically address the effect of adenoviral infusion onmeasures of blood lipids, the adenoviral constructs were furtherpurified to remove any contaminating viral particles and cellulardebris. Another adenoviral experiment was then conducted wherebyAd5-ENHO1 or Ad5-GFPAd5-GFP was injected into the tail vein ofgenetically obese and hyperlipidemic KK-A^(y) mice (n=6 female pergroup). Mice were given an IPGTT 5 days post virus injection, and thensacrificed 2 days later after an overnight fast.

IPGTT revealed that glucose tolerance was not significantly improved byAd5-ENHO1 treatment compared to Ad5-GFPAd5-GFP treatment. Statisticallysignificant reductions in circulating triglycerides (by 28%, p=0.04) andcholesterol (by 20%, p=0.02) were observed in the Ad5-ENHO1 groupcompared to Ad5-GFP controls (Table 1). Liver weight of Ad5-ENHO1 micetended to be lower than liver weight of Ad5-GFP controls (Table 2). Bodyweight, and serum glucose and insulin levels were not significantlydifferent between Ad5-ENHO1 and Ad5-GFP controls (Table 2).

TABLE 3 Characteristics of KK-A^(y) mice treated with Ad5-ENHO1 orAd5-GFP for 8 days. Body Liver Liver as % Weight weight of body GlucoseInsulin Treatment (g) (g) weight (mg/dL) (ng/mL) Ad5-ENHO1 41.9 ± 0.91.8 ± 0.1 4.3 ± 0.1 239 ± 19 4.3 ± 0.7 Ad5-GFP 44.1 ± 1.5 2.1 ± 0.2 4.6± 0.2 246 ± 41 5.1 ± 0.9

ENHO1 mRNA was elevated approximately 4-fold in the liver of KK-A^(y)Ad5-ENHO1-treated mice compared to Ad5-GFP-treated mice (data notshown).

Example 8 Treatment of Obese Insulin Resistant Mice with Ad5-Enho1:Lep^(ob)/Lep^(ob) Mice

To confirm the lipid-lowering effect of adenoviral-mediated ENHO1overexpression, another experiment was performed in which Ad5-ENHO1 orAd5-GFP was injected into the tail vein of Lep^(ob)/Lep^(ob) (OBOB) mice(n=7-8 male per group), another mouse model of hyperlipidemia, steatosisand insulin resistance. The injected mice were not given an IPGTT, butwere monitored for body weight changes, and then sacrificed 7 days postinjection after an overnight fast.

Ad5-GFP-treated mice gained approximately 1 g of body weight over the7-day period. However, Ad5-ENHO1 treatment blocked weight gain in theseobese Lep^(ob)/Lep^(ob) mice (data not shown). Mean body weight was notstatistically different between groups, and food intake was notmeasured. Similar phenotypic changes to that observed in KK-A^(y) mice(See Example 7 above) were also observed. Serum triglycerides werereduced by 40% in the Ad5-ENHO1 group (Ad5-GFP 63±6 mg/dL (Ad5-GFP);38±4 mg/dL (Ad5-ENHO1), p=0.006; Table 2). A 15% reduction in serumcholesterol levels was also observed (173±6 mg/dL (Ad5-GFP); 146±8 mg/dL(Ad5-ENHO1), p=0.02; Table 2). A trend for a reduction in liver weightwas observed that was not significant when expressed as a percentage ofbody weight (p=0.12). Trends for reduced fasting blood glucose andinsulin levels in the Ad5-ENHO1 group did not reach statisticalsignificance.

Purification of the adenoviral constructs and use of larger samplepopulations in the experiments using KK-A^(y) mice and Lep^(ob)/Lep^(ob)mice demonstrated that overexpression of ENHO1 for 7-8 days lead toreduced circulating triglyceride and cholesterol levels compared tocontrol mice. (Table 2) Trends for these effects were observed inprevious experiments using unpurified adenovirus with observations atlater time points. ENHO1 gene expression in the liver was elevated inAd5-ENHO1-treated group in these experiments, whereas no increase wasdetectable in previous experiments at later time points. Without wishingto be bound by this theory, it is believed that increased geneexpression of ENHO1 leads to increased circulating levels of the ENHO1protein.

Example 9 Summary of Results of Treatment of Obese Insulin ResistantMice with Ad5-Enho1

In summary, 5×10⁹ pfu of Ad5-Enho1 or Ad5-GFP was administered into thetail vein of KKAy, B6 Ay/a, or OBOB mice purchased from the JacksonLaboratory (Bar Harbor, Me.). The treatment protocol, shown in FIG. 7C,was based on the pilot experiments demonstrating peak expression ofAd5-Enho1:FLAG during this period (data not shown). Animal and foodweight were recorded daily. Ad5-Enho1 infection was well tolerated inmouse strains used for these experiments. There were no markeddifferences in the body weight (Table 2) of mice infected with Ad5-Enho1compared to controls infected with Ad5-GFP over the treatment period. A4-5 fold increase in Enho1 mRNA was observed in mice infected withAd5-Enho1, compared to Ad5-GFP treated controls (data not shown).

Liver weight and lipid content were consistently reduced in miceinfected with Ad5-Enho1. In KKAy and OBOB mice, significant reductions(≈40%) in fasting triglyceride and total cholesterol were observed. Asmaller decline was observed in Ay/a mice. Meta-analysis of data fromseveral experiments using obese OBOB, KKAy and Ay/a mice mice indicateda 40% reduction of HOMA-IR [(fasting insulin×glucose)/22.5] in OBOB,KKAy and Ay/a mice (Table 4). In Ay/a mice, fasting insulin levels weresignificantly reduced with Ad5-Enho1 (2.6±0.4 vs. 3.9±0.3 ng/ml,P<0.05), with no difference in blood glucose (175±12 vs. 162±11 mg/dL).

TABLE 4 Meta-analysis of HOMA-IR, calculated by mutiplying fastinginsulin and glucose, from OBOB, KKAy and Ay/a mice infected with Ad5-GFPor Ad5-Enho1. Data are expressed as % ± SEM of control group. Ad5-GFPAd5-Enho1 Student's t-test 100 ± 12% 62 ± 4% P < 0.005

Example 10 Overexpression of ENHO1 Effects on Genes Involved in LipidBiosynthesis

Reversal of hepatic steatosis by adipocytokines is at least partiallyattributable to the suppression of hepatic lipogenesis and stimulationof fatty acid oxidation (42,43). To investigate the mechanism by whichEnho1 reduced hepatic lipid content, the expression of genes involved inlipogenesis was measured by quantitative RT-PCR (FIGS. 8A-8D). There wasa coordinate 40-50% reduction in the expression of several genesinvolved in lipogenesis in the liver of OBOB and KKAy mice infected withAd5-Enho1. Enho1 may therefore reduce hepatic lipid content, at least inpart, by inhibiting hepatic lipogenesis.

A significant decrease in fatty acid synthase (Fasn) mRNA in theAd5-ENHO1-treated group was observed compared to the Ad5-GFP controls(FIG. 8A, p=0.02), as well as a decrease in protein levels (data notshown). Fasn mRNA and protein levels were measured using quantitativeRealTime PCR and western blot, as described in D. C. Albarado et al.,“Impaired Coordination of Nutrient Intake and Substrate Oxidation inMelanocortin-4 Receptor Knockout Mice,” Endocrinology, vol. 145, pp.243-252 (2004). Gene expression of the fatty acid translocase CD36 wasalso significantly elevated in the Ad5-ENHO1 group (p=0.02, not shown).FIG. 8A illustrates the expression level of fatty acid synthase (Fasn)mRNA in liver from obese leptin-deficient (Lep^(ob)/Lep^(ob)) mice eightdays after injection with either Ad5-ENHO1 or Ad5-GFP. Data expressed inarbitrary units (AU) (n=6-8/group; “*” p<0.05). Enho1 overexpressioncaused a decrease in Fasn. FIG. 8B illustrates the expression level ofstearoyl-CoA desaturase (Scd1) protein in liver from obeseleptin-deficient (Lep^(ob)/Lep^(ob)) mice eight days after injectionwith either Ad5-ENHO1 or Ad5-GFP. Data expressed in arbitrary units (AU)(n=6-8/group; “*” p<0.05). Again, Enho1 overexpression caused a decreasein Scd1. FIG. 8C illustrates the expression level of acetyl CoAcarboxylase (Acc) mRNA in liver from obese leptin-deficient(Lep^(ob)/Lep^(ob)) mice eight days after injection with eitherAd5-ENHO1 or Ad5-GFP. Data expressed in arbitrary units (AU)(n=6-8/group; “*” p<0.05). Again, the mice with Enho1 showed lowerlevels of liver Acc. FIG. 8D illustrates the expression level of a geneinvolved in insulin resistance (suppressor of cytokine signaling 3 orSocs3) in liver from obese leptin-deficient (Lep^(ob)/Lep^(ob)) miceeight days after injection with either Ad5-ENHO1 or Ad5-GFP. Dataexpressed in arbitrary units (AU) (n=6-8/group; “*” p<0.05). Thepresence of Enho1 again decreased the gene express of Socs3.

Example 11 Transgenic Over Expression of Enho1 (BAP-Enho1)

Transgenic strains over expressing Enho1 were generated using aconstruct (BAP-Enho1) containing the human β-actin promoter, a syntheticexon 1 and intron, and an open reading frame encoding the 76 aa Enho1protein (FIG. 9A; see FIG. 4, the open reading frame is underlined).Eight founders were obtained (2 FVB/NJ, 6 C57BL/6J), with one of the FVBstrains [FVB/NJ.Tg-(BAP-Enho1)AAB20], hereafter referred to as FVB.Tg,exhibiting an increase in hepatic Enho1 expression at 5-6 wk of age(FIG. 9B). FIG. 10 illustrates the results of PCR screening the pups inthe first round of injection of BAP-Enho1 into C57BL/6J oocytes, showingsix pups (6, 8, 14, 19, 21, and 22) with integration of the transgeneinto the genome.

At 5-6 wk of age, reductions in serum TG and TC are already evident inFVB.Tg (Table 5, FIG. 9C). Body weight (Table 5) and FM (FIG. 9D) ofFVB.Tg transgenics was reduced relative to WT littermate. Weight gain ofFVB.Tg mice was also reduced on HFD (60% kJ/fat), indicating protectionfrom diet-induced obesity (FIG. 9E). Reduced body weight and fat contentwas still observed after 1 month on HFD, associated with a tendency forreduced fasting insulin levels of BAP-Enho1 transgenics compared to WTcontrols (180±60 vs. 310±40 pg/ml, 2-tailed Student's t-test P=0.085).

TABLE 5 Data from the first 3 litters of FVB.Tg mice. Body weight Liverweight Liver as % of Glucose Triglyceride Cholesterol (g) (g) bodyweight (mg/dL) (mg/dL) (mg/dL) FVB WT 20.5 ± 1.1  0.88 ± 0.01  4.3 ± 0.4151 ± 14 105 ± 3  219 ± 7  FVB.Tg 17.8 ± 0.1* 0.69 ± 0.01* 3.9 ± 0.2 105± 6* 82 ± 6* 198 ± 4* All mice are female aged approximately 4.5 weeks,n = 3-5 per group. *P < 0.05 compared to control group.

As shown above, it was observed that over expression of Enho1 impairsmetabolic adaptation to fasting, perhaps indicating increased energyexpenditure. To determine whether Enho1 increases energy expenditure,VO2 and RER was measured using indirect calorimetry (15,46,50). ThePennington Biomedical Research Center has a 16 chamber comprehensivelaboratory animal monitoring system (CLAMS) housed in a temperaturecontrolled incubator. The CLAMS simultaneously measured oxygenconsumption (VO2), respiratory exchange ratio (RER, an indicator ofwhole animal substrate oxidation), physical activity in the X and Zaxis, and food intake. Mice were placed in the CLAMS system, and theparameters indicated recorded for 72 h. Mice were fed ad libitum for 48h, with a fast for final 24 h. The results for FVB.Tg are shown in FIGS.13A-13F. Increased weight loss was observed after an overnight fast inobese KKAy, in obese B6 Ay/a mice expressing AdSEnho1 (% weight lossafter overnight fast for Ad5-Enho1 vs Ad5-GFP: 5.5±0.4% vs. 3.5±0.4%,P<0.01), and in 9 wk old lean FVB.Tg (13.6±1.1% vs. 10.0±0.7%, P<0.05).After 1 month of high fat feeding, measurement of energy metabolism byindirect calorimetry indicated significantly increased oxygenconsumption (VO2 in ml/h: BAP-Enho1 Tg 4551±240, WT FVB 3807±145,P<0.05) and physical activity during lights off (X beam breaks per hour:BAP-Enho1 Tg 1431±181; WT FVB 2678±327, P<0.01). Increased energyexpenditure may thus be a factor in the amelioration of diet-inducedobesity and insulin resistance by Enho1.

Example 12 Enho1 Effects Adipocytes

An experiment was conducted to test the use of a recombinant orsynthetic forms of the short secreted polypeptide Enho1³⁴⁻⁷⁶(SEQ IDNO:10). A study was conducted where the response of adipocytes andhepatocytes, two potential sites of Enho1 action, to syntheticEnho1³⁴⁻⁷⁶ was analyzed with changes in extracellular regulated kinase(ERK) as the measured response. ERK1/2 and p38α are important in theregulation of lipolysis and thermogenesis in adipocytes.

Application of 1 μg/ml Enho1³⁴⁻⁷⁶ was associated with a robust increasein the phosphorylation of Erk1 in fully differentiated 3T3-L1 adipocytes(FIG. 11A) and in HepG2 cells (FIG. 11B). These results indicate thatfunctional receptors for Enho1³⁴⁻⁷⁶ are active in adipocytes andhepatocytes, indicating that the synthetic peptide is biologicallyactive. In addition, a shorter, second peptide was synthesized usingamino acids 39 through 76 of SEQ ID NO:2, and called “Enho1³⁹⁻⁷⁶” (SEQID NO:11). Similar results using adipocytes and hepatocytes wereobserved with this peptide. The loss of four amino acids did not appearto affect Enho1 function.

Example 13

Enho1Function in Hypothalamus

Hypothalamic Enho1 may be involved in the regulation of energyhomeostasis. Preliminary analysis using pAB 1 demonstrated the presenceof Enho1-immunoreactivity in neurons located in the arcuate nucleus ofthe hypothalamus (data not shown). It was predicted that a negativecorrelation exists between hypothalamic Enho1 expression and obesity andinsulin resistance. In other words, reduced synthesis of Enho1 in liverand hypothalamus in situations of obesity may be a factor contributingto obesity and insulin resistance.

Enho1 mRNA expression was measured by quantitative RT-PCR in mediobasalhypothalamic blocks from control (low fat diet) and diet-induced obeseC57BL/6J, Mc3rKO and Mc4rKO mice (FIGS. 12A-12B). As predicted, Enho1mRNA abundance in the hypothalamus was reduced in the obese state (FIG.12A). Enho1 expression was also low in mice with elevated HOMA-IRvalues, an indicator of insulin resistance (FIG. 12B).

In contrast, Socs3 mRNA expression was elevated in situations of obesityand insulin resistance (FIG. 11C). This data indicates that reducedsynthesis of Enho1 in situations of obesity is not be limited to theliver. Reduced Enho1 activity in the hypothalamus would also contributeto the development of obesity and insulin resistance.

In summary, an expression vector was constructed using a partial cDNAsequence encoding the predicted 76 residues of ENHO1 (betweennucleotides 207 and 437 of BC021944), with an epitope-tag inserted ontothe carboxyterminal end to allow visualization of the protein on westernblot using a commercially available antibody. Using this construct, itwas verified that the reported sequences encoded a secreted protein.This expression vector and an expression vector encoding the nativeprotein without an epitope label were then used to construct arecombinant adenovirus (Ad5-ENHO1) for use in mouse studies. Injectionsof 5×10⁸ pfu of the adenovirus into the tail vein resulted primarily ininfection of the liver, and to a lesser extent, the spleen. Using theadenovirus expressing the epitope-tagged ENHO1, it was demonstrated that5 days after injection of the virus, there was wide spread distributionof ENHO1 protein in liver, brain, and skeletal muscle. Using thisadenovirus expressing the native form of ENHO1, a new function(reduction of fasting serum triglyceride and cholesterol) was shown forthe secreted protein. A reduction in liver ENHO1 mRNA expression wasobserved in obese mice with hyperlipidemia, with the magnitude of thereduction correlating with the increase of total cholesterol andtriglyceride. Using the adenovirus expressing the native form of ENHO1,it was shown that increasing the expression of the mRNA encoding theENHO1 protein in mouse models of obesity with high cholesterol andtriglycerides was effective at reducing both cholesterol andtriglycerides toward normal levels. The effects of Ad5-ENHO1 infectionto reduce triglyceride and total cholesterol were reproducible, beingobserved in three different mouse strains (Lep^(ob)/Lep^(ob), KKA^(Y),and C57BL/6J). In some mice, an improvement in insulin sensitivity wasobserved in a trend of lower fasting insulin and glucose and animprovement in glucose tolerance test results. The latter observationssuggested that ENHO1 may also be effective at improving insulinsensitivity in an obese, insulin-resistant patient.

The data strongly suggest that full-length ENHO1 peptide (SEQ ID NO: 2),or peptide derivatives, homologues, analogues, or mimetics thereof,delivered by oral intake, injection, subcutaneous patch, or intranasalroutes, could be used as therapeutic or diagnostic agents forhypercholesterolemia, hypertriglyceridemia, insulin resistance, obesity,diabetes, and/or energy imbalance. By methods known in the art,substitutions within the native coding sequence can be made to makederivatives of ENHO1 with increased stability and/or biological potency.Moreover, the ENHO1 peptide can be used to identify its cell receptorwhich can then be used as as-yet-unidentified receptor(s) for ENHO1 is(are) a potential drug target(s) for the development of therapies aimedat reducing total cholesterol, triglycerides, insulin resistance,obesity, diabetes and/or energy imbalance.

A novel secreted protein, Enho1, that is an important factor in theetiology of insulin resistance and hepatic steatosis in the obese statehas been identified by Microarray analysis of gene expression in mousemodels of moderate- to severe-diabesity. Enho1 significantly reducesHOMA-IR, an index of fasting insulin and glucose, and serum lipids inmouse models of type 2 diabetes, and significantly reduces hepaticlipids indicating a reversal of hepatic steatosis. Transgenic overexpression of Enho1 is associated with a lean phenotype. Enho1 may actin a manner similar to leptin and adiponectin, improving metabolicprofile in the obese insulin state by stimulating energy expenditure andincreasing oxidative metabolism.

Example 14 Enho1 Treatment Will Reverse Hepatic Insulin Resistance

Preliminary data from Ad5-Enho1 and BAP-Enho1 transgenics (shown above)indicate improved insulin action and lipid metabolism. In FVB.Tg mice,increased oxidative metabolism is a likely factor explaining theseeffects. It may not be possible to dissect the anti-diabetc actions ofEnho1 from those secondary to reductions of FM. Preliminary data usingrecombinant adenovirus indicate that Enho1 reduces hyperinsulinemia andhyperlipidemia independently of effects on obesity. Further experimentsusing recombinant adenovirus and synthetic peptide may therefore provideimportant information regarding ‘direct’ effects of Enho1 on livermetabolism and insulin sensitivity.

Experiments will be conducted to determine whether the reversal ofhepatic steatosis with Ad-Enho1 treatment is associated with improvedinsulin sensitivity in liver and skeletal muscle by measuring insulinreceptor signaling. These will include analysis of insulin receptorsubstrates 1 and 2 phosphorylation, which are distal to the insulinreceptor tyrosine kinase. Activity of protein kinase B (PKB)/Akt, aserine/threonine kinase involved in regulating glucose transport that isdownstream of phosphatidyl-3′ kinase (44,45), will also be measured asan indicator of insulin receptor activation. Pilot data showingsignificantly greater weight loss of mice infected with Ad5-Enho1suggests increased basal metabolic rate. Whether Ad5-Enho1 increaseswhole body metabolic rate during postprandial and fasting states will bedetermined using indirect calorimetry, and oxidative metabolism intissue lysates from liver, skeletal muscle and brown adipose tissueshall be determined.

The basic protocol for adenovirus treatment is shown in FIG. 7C.Briefly, all mice will have 5×10⁹ pfu of Ad5-ENHO1 or Ad5-GFP injectedinto the tail vein (n=12/group, 24 total). Mice shall be allowed 4 d torecover before use in the following experiments. A liver sample taken attermination will be used to estimate infection by measuring Enho1 mRNAby RealTime PCR and Northern blot analysis. In preliminary experiments,a 4-fold increase in Enho1 mRNA was observed 4-7d post infection. Enho1protein shall also be measured either by Western blot, or through theuse of assays (RIA/ELISA) currently under development. Mice shall beweighed on the day of adenovirus injection, and pre- and post-fasting.If possible, body composition shall be determined using nuclear magneticresonance (NMR) (15,32,46). Mice shall be acclimated to housing inwire-mesh caging that allows for measurement of food intake andspillage, as previously described (15).

If the sexual dimorphic phenotype of the FVB.Tg is reproducible andobserved in BAP-Enho1 transgenics on the C57BL/6J background, the Ad5experiments will be modified to investigate the response of males andfemales. Most of the experiments investigating the response of obeseinsulin resistant mice to Ad5-Enho1 treatment used males; it may be thatfemales will exhibit a different response, perhaps exhibiting weightloss in addition to improved insulin sensitivity. This would not beunprecedented, for example sexual dimorphism has been observed in theresponse of male and female rats to the anorectic actions of insulin andleptin (47,48).

Determine Whether Reversal of Hepatic Steatosis Is Associated WithIncreased Insulin Sensitivity.

This experiment will involve KKAy and Ay/a mice, with two groups oftwelve within genotype (Ad5-Enho1, Ad5-GFP controls) (24 KKAy, 24 Ay/a).On day 5 after an overnight fast, six of the Ad5-ENHO1 and Ad5-GFPgroups shall be administered a single intraperitoneal of insulin (1U/kg), with the remaining six receiving saline. Mice shall be euthanizedeither 10 or 20 minutes (n=3/group) post injection, and tissue samplescollected (liver, quadriceps muscle) and snap frozen on liquid nitrogen.IRS phosphorylation, PKB activity, and FoxO1 phosphorylation shall bemeasured as previously described (15,49). This experiment shall berepeated two more times with only 8 mice per group to do insulin andglucose tolerance tests.

It is predicted that the anti-steatotic effect of Ad5-ENHO1 willincrease insulin-stimulated activity of the insulin receptor tyrosinekinase cascade in liver. Ad5-ENHO1 may also improve insulin signaling inmuscle and/or adipose tissue. The initial studies investigating glucoseclearance resulted in mixed results (data not shown), perhaps due tosub-optimal experimental design [i.e., small numbers of mice (n=4-5);performed at variable times (up two two weeks) after adenovirusinjection]. It is predicted that Ad5-Enho1 will improve glucoseclearance in response to glucose/insulin injections.

Determine Whether Enho1 Increases Whole Body Energy Expenditure andOxidtive Metabolism.

To determine whether hepatic expression of Enho1 increases energyexpenditure, VO2 and RER of obese Ay/a and lean and diet-induced obeseC57BL/6J mice infected with Ad5-Enho1 or Ad5-GFP (n=8/group) shall bemeasured using indirect calorimetry (15,46,50). The PenningtonBiomedical Research Center has a 16 chamber comprehensive laboratoryanimal monitoring system (CLAMS) housed in a temperature controlledincubator. The CLAMS simultaneously measures oxygen consumption (VO2),respiratory exchange ratio (RER, an indicator of whole animal substrateoxidation), physical activity in the X and Z axis, and food intake. Miceshall be placed in the CLAMS system 96 h after adenovirus injection, andthe parameters indicated recorded for 72 h. Mice shall be allowed tofeed ad libitum for 48 h, with a fast for final 24 h. In a separateexperiments, fatty acid oxidation (C¹⁴-palmitate) and mitochondrialenzyme function (citrate synthase activity, cytochrome c oxidase) willbe measured in liver, gastrocnemius, and brown adipose tissue collectedfrom Ay/a and C57BL/6J mice infected with Ad5-Enho1 or Ad5-GFP(n=8/group). A portion of liver shall be collected and snap frozen formeasurement of mRNA and protein expression of transcription factors(i.e., SREBP1c, PPARγ) and enzyme involved in lipogenesis (32).

The increased weight loss of mice infected with Ad5-Enho1 indicatesincreased basal metabolic rate, a finding corroborated by fasting weightloss and indirect calorimetry data from BAP-Enho1 transgenics. (SeeFIGS. 13A-F) It is predicted that infection with Ad5-Enho1 will increaseVO2, although this may only be evident during the fasting phase. Anincrease in mitochondrial oxidative enzyme activity in liver only wouldbe consistent with Enho1 acting as an autocrine/paracrine factor.Increased mitochondrial oxidative enzyme activity in skeletal muscle andbrown adipose tissue would suggest an endocrine function, either actingthrough the autonomic nervous system or through receptors expressed inmuscle and/or brown adipose tissue. If Ad5-Enho1 is markedly increasingenergy expenditure (as observed in FVB.Tg) but is not affecting bodyweight, then a compensatory increase in food intake would be predicted.A reduction in the expression of genes involved in lipogenesis, asobserved in OBOB mice treated with Ad5-Enho1, is also predicted. (FIG.8A-8D).

Example 15 Transgenic Expression of Enho1 Prevents Obesity and InsulinResistance

Transgenic mice over expressing leptin (51,52) and adiponectin (53,54)have demonstrated the anti-diabetic action of over expression of eitherprotein. It is expected that over expression of Enho1 will have outcomescomparable to that observed with over expression of adiponectin andleptin; i.e. improved insulin sensitivity and glucose tolerance, andlower fasting lipids in situations of diet- and genetically inducedobesity. It is also predicted that Enho1 will increases energyexpenditure by increasing oxidative metabolism in liver, skeletalmuscle, and/or brown adipose tissue. A comprehensive analysis of thephysiology of Enho1 action is important for future experiments thatfocus on the mechanism(s) by which this polypeptide regulates energymetabolism and insulin signaling.

Transgenics:

A sequence encoding the 76 aa protein has been ligated into a synthetictransgene controlled by the human β-actin promoter (BAP) (FIG. 9A).Enho1 is a secreted polypeptide (FIG. 4), and thus tissue-selectivity isnot important for these transgenic studies. Promoters specific fortissues have not been used where the endogenous gene is abundantlyexpressed, because suppression of the endogenous gene may limit efficacyof over expression (53,54). A comprehensive analysis of mRNA expressionshall be completed using quantitative RT-PCR (qRT-PCR) as previouslydescribed (31,32,46) and Northern blot analysis. Protein levels shall bemeasured by Western blot using the two polyclonal antibodies against theN- and C-terminus of Enho³⁴⁻⁷(pAB1, pAB2) (FIG. 5). This may requireusing immunoprecitipitation to detect protein. Alternatively, ifantibodies in hand or in development are useful for developing sensitiveand quantitative assays, then these shall be used. Transgene copy numbershall be determined by Southern blot analysis. A sub-aim of thisexperiment is therefore a more comprehensive analysis of Enho1 mRNAexpression in mouse tissues (liver, hypothalamus, forebrain, hindbrain,skeletal and cardiac muscle, retroperitoneal and inquinal adiposedepots, stomach, intestine, pancreas, kidney). Major organs (heart,kidney, gut, liver) shall be weighed and inspected histologically formajor morphological changes.

We have shown that Enho1 is one of a small group of secretedpolypeptides (leptin, adiponectin) that, when over expressed, improvesmetabolic profile (i.e. increased insulin sensitivity, reduced hepaticlipogenesis) in mouse models of obesity and insulin resistance. The overexpression of Enho1 has leptin-like effects on energy metabolism,protecting against diet-induced obesity and insulin resistance.Administering Enho1 can reverse insulin resistance and dyslipidemiaassociated with diet- and genetically-induced obesity, and can preventor delay onset of diabesity.

Examples 16 to 25 were carried out using human Enho1, a 76 amino acidpeptide having the sequence:

(SEQ ID NO: 14) MGAAISQGALIAIVCNGLVGFLLLLLWVILCWACHSRSADVDSLSESSPNSSPGPCPEKAPPPQKPSHEGSYLLQP

Statistical analysis of the results was carried out using ANOVA analysistools with post hoc tests. Data are reported as the mean±standard error(SE).

Of course, the skilled artisan would know and realize that experimentssimilar to those described herein may be carried out to determine theeffect of Enho-1 upon various parameters of metabolism such as weightloss, hepatic steatosis, lipids and triglycerides, glucose, insulinlevels and the like. For example, the length of treatment time may bevaried, the genetic make up of the mice or rat strains may be different,different diet regimes may be applied, etc. These experiments are in noway intended to be binding and are representative of those which areuseful to study the effects of peptides such as Enho-1 in mammals.

Example 16 Effect of Enho1 on Weight Loss (Short-Term Treatment)

The effect of Enho-1 upon weight loss was tested over the course of 3days using KKAy mice. The mice were fed Breeder Chow (Purina 5015) andwere approximately 12-14 weeks of age at the start of the experiment.Six control mice received vehicle only while six test mice each receiveddoses of either 900 nmol/kg/d or 9000 nmol/kg/d. The injections weregiven as 3 intraperitoneal (ip) injections at 0600, 1400 and 2000 h ondays 1 and 2; over the 3 day period, the mice received a total of 7injections. All animals were euthanized 5 hours after the last injectiongiven the morning of the third day. Food was removed after the finalinjection.

The six control mice had a mean pre-treatment weight of 35.3 g (SE±1.2g) and a mean post-treatment weight of 33.6 g (SE±1.0). The differenceof −1.7 g (SE±0.3) represents a −4.8% (SE±0.6) reduction in weight.

The six test mice which received 900 nmol/kg/d of Enho1 had apre-treatment weight of 35.3 g (SE±1.1) and post-treatment weight of33.0 g (SE±1.0). The difference of −2.3 g (SE±0.2) represents a −6.5%(SE±0.6) reduction in weight.

The six test mice which received 9000 nmol/kg/d of Enho1 had apre-treatment weight of 35.6 g (SE±0.6) and post-treatment weight of33.1 g (SE±0.4). The difference of −2.6 g (SE±0.3) represents a −7.1%(SE±0.8) reduction in weight.

These results are reported in Table 6.

TABLE 6 the effect of Enho-1 upon weight loss 900 9000 Control nmol/kg/dnmol/kg/d Weight  35.3 ± 1.2 g 35.3 ± 1.1 35.6 ± 0.6 (pre-treatment)Weight 33.6 ± 1.0 33.0 ± 1.0 33.1 ± 0.4 (termination) Delta body weightGrams −1.7 ± 0.3 −2.3 ± 0.2 −2.6 ± 0.3 % −4.8 ± 0.6 −6.5 ± 0.6 −7.1 ±0.8 P = 0.073 ANOVA

Example 17 Effect of Enho1 on Insulin and Glucose Levels

The effect of Enho-1 upon insulin and glucose was tested over the courseof 3 days using KKAy mice. The mice were fed Breeder Chow (Purina 5015)and were approximately 12-14 weeks of age at the start of theexperiment. Six control mice received vehicle only while five or sixtest mice each received doses of either 900 nmol/kg/d or 9000 nmol/kg/d,respectively. The injections were given as 3 ip injections at 0600, 1400and 2000 h on days 1 and 2; over the 3 day period, the mice received atotal of 7 injections. All animals were euthanized 5 hours after thelast injection given the morning of the third day. Food was removedafter the final injection. The HOMA-IR for each treatment was calculatedusing the following formula:

((glucose mg/dL÷18)×(insulin ng/ml×25.05))÷22.5

The six control mice demonstrated a post-test mean blood glucose levelof 524 mg/dL (SE±48) and an insulin level of 7.4 ng/ml (SE±0.6); theHOMA-IR value for the control group was determined to be 234 (SE±16).

The five test mice which received 900 nmol/kg/d of Enho1 demonstrated apost-test mean blood glucose level of 474 mg/dL (SE±29) and an insulinlevel of 5.7 ng/ml (SE±0.5); the HOMA-IR value for this group wasdetermined to be 167 (SE±18), representing a decrease of 29% as comparedto the control group. The blood glucose levels decrease by approximately10% while the insulin levels were reduced by about 22%.

The six test mice which received 9000 nmol/kg/d of Enho1 demonstrated apost-test mean blood glucose level of 480 mg/dL (SE±38) and an insulinlevel of 7.0 ng/ml (SE±0.8); the HOMA-IR value for this group wasdetermined to be 213 (SE±37), representing a decrease of 9% as comparedto the control group.

These results are reported in Table 7.

TABLE 7 the effect of Enho-1 upon insulin and glucose 900 9000 Controlnmol/kg/d nmol/kg/d Blood glucose 524 ± 48 474 ± 29 480 ± 38 (mg/dL)Insulin  7.4 ± 0.6  5.7 ± 0.5  7.0 ± 0.8 (ng/ml) HOMA-IR 234 ± 16 167 ±18 213 ± 37 (↓ 29%) (↓ 9%)

Example 18 Effect of Enho1 on Hepatic Steatosis

The effect of Enho-1 upon liver weight, liver lipid content and liver TGwas tested over the course of 3 days using KKAy mice. The mice were fedBreeder Chow (Purina 5015) and were approximately 12-14 weeks of age atthe start of the experiment. Six control mice received vehicle onlywhile five or six test mice each received doses of either 900 nmol/kg/dor 9000 nmol/kg/d, respectively. The injections were given as 3 ipinjections at 0600, 1400 and 2000 h on days 1 and 2; over the 3 dayperiod, the mice received a total of 7 injections. All animals wereeuthanized 5 hours after the last injection given the morning of thethird day. Food was removed after the final injection.

The six control mice exhibited a mean liver weight of 1.6 g (SE±0.1),representing 4.6% (SE±0.2) of total body weight. The mean liver lipidlevel for the control group was 94 mg/g (SE±6), with total lipidsmeasuring 147 mg/g (SE±14). Liver TG for the control group wasdetermined to be 59 mg/g (SE±5; P<0.05) while the total liver TG wasdetermined to be 92 mg/g (SE±10; p=0.062).

The five test mice which received 900 nmol/kg/d of Enho1 demonstrated apost-test mean liver weight of 1.6 g (SE±0.1), representing 5.0%(SE±0.2) of body weight. The mean liver lipid level for this group wasdetermined to be 82 mg/g (SE±9), with total lipids measuring 137 mg/g(SE±18). Liver TG for this group was determined to be 40 mg/g (SE±8;P<0.05) while the total liver TG was determined to be 66 mg/g (SE±13;p=0.062).

The six test mice which received 9000 nmol/kg/d of Enho1 demonstrated apost-test mean liver weight of 1.6 g (SE±0.1), representing 4.9%(SE±0.3) of body weight. The mean liver lipid level for this group wasdetermined to be 87 mg/g (SE±6), with total lipids measuring 143 mg/g(SE±17). Liver TG for this group was determined to be 32 mg/g (SE±5;P<0.05) while the total liver TG was determined to be 53 mg/g (SE±11;p=0.062).

These data show that Enho1 is capable of inducing a dose-dependentreversal of hepatic steatosis. In mice receiving the 900 nmol/kg/d andthe 9000 nmol/kg/d dosage, the livers, the TG was reduced by 32% and46%, respectively, as compared to the control group.

These results are reported in Table 8.

TABLE 8 the effect of Enho-1 upon liver weight, liver lipid content andliver TG 900 9000 Control nmol/kg/d nmol/kg/d Liver weight (g) 1.6 ± 0.11.6 ± 0.1 1.6 ± 0.1 % body weight 4.6 ± 0.2 5.0 ± 0.2 4.9 ± 0.3 Liverlipid mg/g 94 ± 6  82 ± 9  87 ± 6  total 147 ± 14  137 ± 18  143 ± 17 Liver TG mg/g 59 ± 5  40 ± 8  32 ± 5  P < 0.05 ANOVA total P < 0.05 vscontrol P = 0.062 ANOVA 92 ± 10 66 ± 13 53 ± 11

Example 19 Effect of Enho1 on Weight Loss (Short Term) in Obese Mice

The effect of Enho-1 upon weight loss was tested over the course of 3days using diet induced obese (DIO) C57BL/6J mice (Jackson Laboratories,Bar Harbor, Me.). The mice were fed Research Diets 12492 (60% kJ/fat)for 12 weeks, resulting in obesity and moderate hyperglycemia in theanimals. The mice were approximately 20-22 weeks of age and 30-50 g inweight at the start of the experiment. Fasting blood glucose wasdetermined to be approximately 170-220 mg/dL. Six control mice receivedvehicle only while six test mice each received doses of either 90nmol/kg/d, 900 nmol/kg/d or 9000 nmol/kg/d. The injections were given as3 ip injections at 0600, 1400 and 2000 h on days 1 and 2; over the 3 dayperiod, the mice received a total of 7 injections. All animals wereeuthanized 5 hours after the last injection given the morning of thethird day. Food was removed after the final injection.

The six control mice exhibited mean pre- and post-treatment weights of42.7 g (SE±2.4 g) and 41.7 g (SE±2.3), respectively, representing a 2.4%(SE±0.5) reduction in total body weight.

The six test mice receiving 90 nmol/kg/d Enho1 exhibited mean pre- andpost-treatment weights of 42.9 g (SE±1.2) and 41.7 g (SD±0.9),respectively, representing a 2.9% (SE±0.6) reduction in total bodyweight.

The six test mice receiving 900 nmol/kg/d Enho1 exhibited mean pre- andpost-treatment weights of 42.8 g (SE±2.9) and 41.3 g (SE±2.7),respectively, representing a 3.5% (SE±0.7) reduction in total bodyweight.

The six test mice receiving 9000 nmol/kg/d Enho1 exhibited mean pre- andpost-treatment weights of 42.7 g (SE±1.6) and 40.9 g (SE±1.6),respectively, representing a 4.1% (SE±0.6) reduction in total bodyweight.

These results are reported in Table 9.

TABLE 9 the effect of Enho-1 upon weight loss 90 900 9000 Controlnmol/kg/d nmol/kg/d nmol/kg/d Weight 42.7 ± 2.4 42.9 ± 1.2 42.8 ± 2.942.7 ± 1.6 (pre-treatment) Weight 41.7 ± 2.3 41.7 ± 0.9 41.3 ± 2.7 40.9± 1.6 (termination) Delta body weight Grams −1.0 ± 0.2 −1.3 ± 0.3 −1.5 ±0.3 −1.7 ± 0.2 % −2.4 ± 0.5 −2.9 ± 0.6 −3.5 ± 0.7 −4.1 ± 0.6

Example 20 Effect of Enho1 on Insulin and Glucose Levels

The effect of Enho-1 upon insulin and glucose was tested over the courseof 3 days using diet induced obese (DIO) C57BL/6J mice (JacksonLaboratories, Bar Harbor, Me.). The mice were fed Research Diets 12492(60% kJ/fat) for 12 weeks, resulting in obesity and moderatehyperglycemia in the animals. The mice were approximately 20-22 weeks ofage and 30-50 g in weight at the start of the experiment. Fasting bloodglucose was determined to be approximately 170-220 mg/dL. Six controlmice received vehicle only while six test mice each received doses ofeither 90 nmol/kg/d, 900 nmol/kg/d or 9000 nmol/kg/d. The injectionswere given as 3 ip injections at 0600, 1400 and 2000 h on days 1 and 2;over the 3 day period, the mice received a total of 7 injections. Allanimals were euthanized 5 hours after the last injection given themorning of the third day. Food was removed after the final injection.

The HOMA-IR for each treatment was calculated using the followingformula:

((glucose mg/dL±18)×(insulin ng/ml×25.05))±22.5

The six control mice demonstrated a post-test mean blood glucose levelof 196 mg/dL (SE±7) and an insulin level of 4.8 ng/ml (SE±0.2); theHOMA-IR value for the control group was determined to be 59 (SE±3;p=0.065).

The six test mice which received 90 nmol/kg/d of Enho1 demonstrated apost-test mean blood glucose level of 204 mg/dL (SE±7) and an insulinlevel of 3.8 ng/ml (SE±0.4); the HOMA-IR value for this group wasdetermined to be 50 (SE±5), representing a decrease of 15% as comparedto the control group.

The six test mice which received 900 nmol/kg/d of Enho1 demonstrated apost-test mean blood glucose level of 166 mg/dL (SE±13; P>0.05) and aninsulin level of 4.2 ng/ml (SE±0.5); the HOMA-IR value for this groupwas determined to be 42 (SE±3), representing a decrease of 29% (p<0.02)as compared to the control group. The blood glucose levels decreased byapproximately 15% while the insulin levels were reduced by about 14%.

The six test mice which received 9000 nmol/kg/d of Enho1 demonstrated apost-test mean blood glucose level of 198 mg/dL (SE±9) and an insulinlevel of 4.2 ng/ml (SE±0.3); the HOMA-IR value for this group wasdetermined to be 52 (SE±4), representing a decrease of 12% as comparedto the control group.

These results are reported in Table 10.

TABLE 10 the effect of Enho-1 upon insulin and glucose 90 900 9000Control nmol/kg/d nmol/kg/d nmol/kg/d Blood glucose 196 ± 7  204 ± 7 166 ± 13 198 ± 9  (mg/dL) P < 0.05 vs 90 P < 0.05 ANOVA Insulin  4.8 ±0.2  3.8 ± 0.4  4.2 ± 0.5  4.2 ± 0.3 (ng/ml) HOMA-IR 59 ± 3 50 ± 5 42 ±3 52 ± 4 1-way ANOVA (↓ 15%) (↓ 29%) (↓ 12%) p = 0.065

Example 21 Effect of Enho1 on Hepatic Steatosis

The effect of Enho-1 upon liver weight, liver lipid content and liver TGwas tested over the course of 3 days using diet induced obese (DIO)C57BL/6J mice (Jackson Laboratories, Bar Harbor, Me.). The mice were fedResearch Diets 12492 (60% kJ/fat) for 12 weeks, resulting in obesity andmoderate hyperglycemia in the animals. The mice were approximately 20-22weeks of age and 30-50 g in weight at the start of the experiment.Fasting blood glucose was determined to be approximately 170-220 mg/dL.Six control mice received vehicle only while six test mice each receiveddoses of either 90 nmol/kg/d, 900 nmol/kg/d or 9000 nmol/kg/d. Theinjections were given as 3 ip injections at 0600, 1400 and 2000 h ondays 1 and 2; over the 3 day period, the mice received a total of 7injections. All animals were euthanized 5 hours after the last injectiongiven the morning of the third day. Food was removed after the finalinjection.

The six control mice exhibited a mean liver weight of 1.6 g (SE±0.2),representing 3.7% (SE±0.2) of total body weight. The mean liver lipidlevel for the control group was 155 mg/g (SE±39), with total lipidsmeasuring 272 mg/g (SE±89). Liver TG for the control group wasdetermined to be 58 mg/g (SE±11) while the total liver TG was determinedto be 87 mg/g (SE±15).

The six test mice which received 90 nmol/kg/d of Enho1 demonstrated apost-test mean liver weight of 1.5 g (SE±0.0), representing 3.6%(SE±0.1) of body weight. The mean liver lipid level for this group wasdetermined to be 126 mg/g (SE±9), with total lipids measuring 188 mg/g(SE±13). Liver TG for this group was determined to be 57 mg/g (SE±8)while the total liver TG was determined to be 86 mg/g (SE±14).

The six test mice which received 900 nmol/kg/d of Enho1 demonstrated apost-test mean liver weight of 1.5 g (SE±0.2), representing 3.5%(SE±0.2) of body weight. The mean liver lipid level for this group wasdetermined to be 137 mg/g (SE±34), with total lipids measuring 226 mg/g(SE±77). Liver TG for this group was determined to be 48 mg/g (SE±12)while the total liver TG was determined to be 66 mg/g (SE±14).

The six test mice which received 9000 nmol/kg/d of Enho1 demonstrated apost-test mean liver weight of 1.4 g (SE±0.2), representing 3.4%(SE±0.3) of body weight. The mean liver lipid level for this group wasdetermined to be 139 mg/g (SE±42), with total lipids measuring 230 mg/g(SE±96). Liver TG for this group was determined to be 47 mg/g (SE±9)while the total liver TG was determined to be 63 mg/g (SE±10).

These data show that Enho1 is capable of inducing a dose-dependentreversal of hepatic steatosis. In mice receiving the 9000 nmol/kg/ddosage, the livers, the liver TG was reduced by 18-19% while serum TGwas reduced by 20% (p<0.05; 1-tailed t-test) as compared to the controlgroup.

These results are reported in Table 11.

TABLE 11 The effect of Enho-1 upon liver weight, liver lipid content andliver TG 90 900 9000 Control nmol/kg/d nmol/kg/d nmol/kg/d Liver weightgrams 1.6 ± 0.2 1.5 ± 0.0 1.5 ± 0.2 1.4 ± 0.2 % body 3.7 ± 0.2 3.6 ± 0.13.5 ± 0.2 3.4 ± 0.3 weight Liver lipid mg/g 155 ± 39  126 ± 8  137 ± 34 139 ± 42  total 272 ± 89  188 ± 13  226 ± 77  230 ± 96  Liver TG mg/g 58± 11 57 ± 8  48 ± 12 47 ± 9  total 87 ± 14 86 ± 14 66 ± 14 63 ± 10

Example 22 Effect of Enho1 on Serum Lipids

The effect of Enho-1 upon liver serum lipid content was tested over thecourse of 8 days using obese (ob/ob) mice. The mice were fed ResearchDiets 12450 and were approximately 9-10 weeks of age at the start of theexperiment. Control mice received vehicle only while test mice eachreceived doses of either 90 nmol/kg/d, 900 nmol/kg/d or 9000 nmol/kg/d.The injections were given as 3 ip injections at 0800, 1200 and 1600 hfor 7 days. On day 8, the mice were given the 0800 h injection and weresacrificed 1-3 h later, at which time blood samples were taken.

Blood glucose levels were measured using an Accu-Chek glucometer.Insulin levels were measured by ELISA (Mercodia Mouse Insulin ELISA,ALPCO) while triglycerides were measured using a Triglyceride L-Type TGH kit (Wako Diagnostics).

Eight control mice exhibited a mean blood triglyceride level of 29.6mg/dL (SE±5.9).

Eight test mice which received 90 nmol/kg/d of Enho1 demonstrated apost-test blood TG level of 19.4 mg/dL (SE±2.2), a decrease of 34% incomparison with the control.

Seven test mice which received 900 nmol/kg/d of Enho1 demonstrated apost-test blood TG level of 18.4 mg/dL (SE±2.3), a decrease of 38% fromthe control.

The final group of seven mice which received 9000 nmol/kg/d of Enho1demonstrated a post-test blood TG level of 22.0 mg/dL (SE±2.2), adecrease of 26% from the control.

These results are reported in Table 12.

TABLE 12 The effect of Enho-1 upon liver serum lipid content 90 900 9000Control nmol/kg/d nmol/kg/d nmol/kg/d TG 29.6 ± 5.9 19.4 ± 2.2 18.4 ±2.3 22.0 ± 2.2 (mg/dL) (↓ 34%) (↓ 38%) (↓ 26%)

Example 23 Effect of Enho1 on Weight Loss

The effect of Enho-1 upon weight loss was tested over the course of 7days using DIO C57BL/6J mice (Charles River Laboratories). The mice werefed a high fat diet (Research Diets 12492) for 10 weeks prior to thestart of the experiment, which was begun when the mice were 15-16 weeksof age. Control mice received vehicle only while test mice each receiveddoses of either 90 nmol/kg/d, 900 nmol/kg/d or 9000 nmol/kg/d. Theinjections were given as 3 ip injections at 0800, 1200 and 1600 h for 7days. On day 8, the mice were given the 0800 h injection and weresacrificed 1-3 h later.

The eight control mice exhibited mean pre- and post-treatment weights of34.2 g (SE±1.5 g) and 32.6 g (SE±1.0), respectively, representing a 4.5%(SE±1.5) reduction in total body weight.

Eight test mice receiving 90 nmol/kg/d Enho1 exhibited mean pre- andpost-treatment weights of 36.5 g (SE±1.3) and 34.0 g (SD±0.9),respectively, representing a 6.6% (SE±1.1) reduction in total bodyweight.

Seven test mice receiving 900 nmol/kg/d Enho1 exhibited mean pre- andpost-treatment weights of 37.9 g (SE±1.4) and 36.0 g (SE±1.2),respectively, representing a 7.1% (SE±1.2) reduction in total bodyweight.

Lastly, seven test mice receiving 9000 nmol/kg/d Enho1 exhibited meanpre- and post-treatment weights of 33.8 g (SE±10.8) and 31.6 g (SE±0.7),respectively, representing a 6.7% (SE±1.3) reduction in total bodyweight.

These results are reported in Table 13.

TABLE 13 The effect of Enho-1 upon weight loss 90 900 9000 Controlnmol/kg/d nmol/kg/d nmol/kg/d Weight 34.2 ± 1.5 36.5 ± 1.3 37.9 ± 1.433.8 ± 0.8 (pre-treatment) Weight 32.6 ± 1.0 34.0 ± 1.0 36.0 ± 1.2 31.6± 0.7 (termination) Delta body weight Grams −1.6 ± 0.6 −2.5 ± 0.4 −2.8 ±0.6 −2.3 ± 0.5 % −4.5 ± 1.5 −6.6 ± 1.1 −7.1 ± 1.2 −6.7 ± 1.3

Example 24 Effect of Enho1 on Insulin and Glucose

The effect of Enho-1 upon insulin and glucose was tested over the courseof 7 days using DIO C57BL/6J mice (Charles River Laboratories). The micewere fed a high fat diet (Research Diets 12492) for 10 weeks prior tothe start of the experiment, which was begun when the mice were 15-16weeks of age. Control mice received vehicle only while test mice eachreceived doses of either 90 nmol/kg/d, 900 nmol/kg/d or 9000 nmol/kg/d.The injections were given as 3 ip injections at 0800, 1200 and 1600 hfor 7 days. On day 8, the mice were given the 0800 h injection and weresacrificed 1-3 h later at which time blood samples were collected andlevels of insulin and glucose determined.

The HOMA-IR for each treatment was calculated using the followingformula:

((glucose mg/dL÷18)×(insulin ng/ml×25.05))÷22.5

Eight control mice demonstrated a post-test mean blood glucose level of191 mg/dL (SE±10) and an insulin level of 1.9 ng/ml (SE±0.3); theHOMA-IR value for the control group was determined to be 22 (SE±4).

The eight test mice which received 90 nmol/kg/d of Enho1 demonstrated apost-test mean blood glucose level of 182 mg/dL (SE±17) and an insulinlevel of 1.3 ng/ml (SE±0.1); the HOMA-IR value for this group wasdetermined to be 15 (SE±2), representing a decrease of 15% as comparedto the control group.

The seven test mice which received 900 nmol/kg/d of Enho1 demonstrated apost-test mean blood glucose level of 212 mg/dL (SE±11) and an insulinlevel of 2.2 ng/ml (SE±0.3); the HOMA-IR value for this group wasdetermined to be 29 (SE±4), representing a decrease of 29% (p<0.02) ascompared to the control group.

The seven test mice which received 9000 nmol/kg/d of Enho1 demonstrateda post-test mean blood glucose level of 190 mg/dL (SE±16) and an insulinlevel of 1.3 ng/ml (SE±0.2); the HOMA-IR value for this group wasdetermined to be 16 (SE±4), representing a decrease of 27 as compared tothe control group.

These results are reported in Table 14.

TABLE 14 The effect of Enho-1 upon insulin and glucose 90 900 9000Control nmol/kg/d nmol/kg/d nmol/kg/d Blood glucose 191 ± 10 182 ± 17212 ± 11 190 ± 16 (mg/dL) Insulin  1.9 ± 0.3  1.3 ± 0.1  2.2 ± 0.3  1.3± 0.2 (ng/ml) HOMA-IR 22 ± 4 15 ± 2 29 ± 4 16 ± 4 (↓ 32%) (↓ 27%)

Example 25 Effect of Enho1 on Blood Lipids

The effect of Enho-1 upon levels of blood lipids was tested over thecourse of 7 days using DIO C57BL/6J mice (Charles River Laboratories).The mice were fed a high fat diet (Research Diets 12492) for 10 weeksprior to the start of the experiment, which was begun when the mice were15-16 weeks of age. Control mice received vehicle only while test miceeach received doses of either 90 nmol/kg/d, 900 nmol/kg/d or 9000nmol/kg/d. The injections were given as 3 ip injections at 0800, 1200and 1600 h for 7 days. On day 8, the mice were given the 0800 hinjection and were sacrificed 1-3 h later at which time blood sampleswere collected and levels of trigylcerides determined. Triglycerideswere measured using a Triglyceride L-Type TG H kit (Wako Diagnostics).

Eight control mice exhibited a mean blood triglyceride level of 22.9mg/dL (SE±2.8).

Eight test mice which received 90 nmol/kg/d of Enho1 demonstrated apost-test blood TG level of 22.5 mg/dL (SE±6.0).

Seven test mice which received 900 nmol/kg/d of Enho1 demonstrated apost-test blood TG level of 38.0 mg/dL (SE±12.5).

The final group of seven mice which received 9000 nmol/kg/d of Enho1demonstrated a post-test blood TG level of 18.5 mg/dL (SE±1.7), adecrease of 19% from the control.

These results are reported in Table 15.

TABLE 15 The effect of Enho-1 upon levels of blood lipids 90 900 9000Control nmol/kg/d nmol/kg/d nmol/kg/d TG 22.9 ± 2.8 22.5 ± 6.0 38.0 ±12.5 18.5 ± 1.7 (mg/dL) (↓ 19%)

Miscellaneous

An “effective amount” of a Enho1 protein or peptide is an amount thatdecreases the level of insulin resistance or of dyslipidemia, or thatprevents, delays or reduces the incidence of the onset of type 2diabetes in obese insulin resistant patients by a statisticallysignificant degree. “Statistical significance” is determined as theP<0.05 level, or by such other measure of statistical significance as iscommonly used in the art for a particular type of experimentaldetermination.

The term “Enho1” used herein and in the claims refers to the proteinEnho1 (SEQ. ID. NO. 2), its functional peptides (e.g., Enho1³⁴⁻⁷⁶),derivatives and analogs. The terms “derivatives” and “analogs” areunderstood to be proteins that are similar in structure to Enho1 andthat exhibit a qualitatively similar effect to the unmodified Enho1. Theterm “functional peptide” refers to a piece of the Enho1 protein thatstill binds to the Enho1 receptor or is able to activate changes insidebody cells, e.g., adipocytes or hepatocytes.

The administration of Enho1, its functional peptides, its analogs andderivatives in accordance with the present invention may be used toreverse insulin resistance and dyslipidemia, to delay onset to type 2diabetes in obese insulin resistant subjects, and to prevent or delayonset of obesity. These compounds can also be used as therapeutic ordiagnostic agents for hypercholesterolemia, hypertriglyceridemia,insulin resistance, obesity, and diabetes.

The term “therapeutically effective amount” as used herein refers to anamount of Enho1 protein, a fragment, or a derivative or analogsufficient either increase body energy expenditure, decrease serumtriglyceride, decrease serum cholesterol, decrease hyperlipidemia, ordecrease insulin resistance to a statistically significant degree(p<0.05). The dosage ranges for the administration of Enho1 protein arethose that produce the desired effect. Generally, the dosage will varywith the age, weight, condition, sex of the patient. A person ofordinary skill in the art, given the teachings of the presentspecification, may readily determine suitable dosage ranges. The dosagecan be adjusted by the individual physician in the event of anycontraindications. In any event, the effectiveness of treatment can bedetermined by monitoring either the body metabolism, body weight, or theserum glucose, triglyceride, cholesterol levels by methods well known tothose in the field. Moreover, Enho1 can be applied in pharmaceuticallyacceptable carriers known in the art.

This method of treatment may be used in vertebrates generally, includinghuman and non-human mammals. Peptides in accordance with the presentinvention may be administered to a patient by any suitable means,including oral, intravenous, parenteral, subcutaneous, intrapulmonary,and intranasal administration.

Parenteral infusions include intramuscular, intravenous, intraarterial,or intraperitoneal administration. The compound may also be administeredtransdermally, for example in the form of a slow-release subcutaneousimplant, or orally in the form of capsules, powders, or granules. It mayalso be administered by inhalation.

Pharmaceutically acceptable carrier preparations for parenteraladministration include sterile, aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. The active therapeutic ingredient maybe mixed with excipients that are pharmaceutically acceptable and arecompatible with the active ingredient. Suitable excipients includewater, saline, dextrose, glycerol and ethanol, or combinations thereof.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, inertgases, and the like.

The form may vary depending upon the route of administration. Forexample, compositions for injection may be provided in the form of anampule, each containing a unit dose amount, or in the form of acontainer containing multiple doses.

The compound may be formulated into therapeutic compositions aspharmaceutically acceptable salts. These salts include acid additionsalts formed with inorganic acids, for example hydrochloric orphosphoric acid, or organic acids such as acetic, oxalic, or tartaricacid, and the like. Salts also include those formed from inorganic basessuch as, for example, sodium, potassium, ammonium, calcium or ferrichydroxides, and organic bases such as isopropylamine, trimethylamine,histidine, procaine and the like. The compositions may be administeredintravenously, subcutaneously, intramuscularly.

Controlled delivery may be achieved by admixing the active ingredientwith appropriate macromolecules, for example, polyesters, polyaminoacids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, prolamine sulfate, or lactide/glycolidecopolymers. The rate of release of the active compound may be controlledby altering the concentration of the macromolecule.

Another method for controlling the duration of action comprisesincorporating the active compound into particles of a polymericsubstance such as a polyester, peptide, hydrogel, polylactide/glycolidecopolymer, or ethylenevinylacetate copolymers. Alternatively, an activecompound may be encapsulated in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,by the use of hydroxymethylcellulose or gelatin-microcapsules orpoly(methylmethacrylate) microcapsules, respectively, or in a colloiddrug delivery system. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes.

In addition, all or portions of the nucleic acid sequence (SEQ ID NO:1)(e.g., the open reading frame underlined in FIG. 4) can be used to makeplasmids or vectors to incorporate the Enho1 gene into organisms toincrease the production of the Enho1 protein. It will be understood bythose skilled in the art that the nucleic acid sequence of SEQ ID NO:1is not the only sequence that can be used to produce the Enho1 protein.Also contemplated are those nucleic acid sequences that encode identicalproteins but that, because of the degeneracy of the genetic code,possess different nucleotide sequences. The genetic code may be found innumerous references concerning genetics or biology, including, forexample, FIG. 9.1 on page 214 of B. Lewin, Genes VI (Oxford UniversityPress, New York, 1997). FIG. 9.3 on page 216 of Lewin directlyillustrates the degeneracy of the genetic code. For example, the codonfor asparagine may be AAT or AAC.

The invention also encompasses nucleotide sequences encoding Enho1proteins having one or more silent amino acid changes in portions of themolecule not involved with receptor binding or protein secretion. Forexample, alterations in the nucleotide sequence that result in theproduction of a chemically equivalent amino acid at a given site arecontemplated; thus, a codon for the amino acid alanine, a hydrophobicamino acid, may be substituted by a codon encoding another hydrophobicresidue, such as glycine, or may be substituted with a more hydrophobicresidue such as valine, leucine, or isoleucine. Similarly, changes thatresult in the substitution of one negatively-charged residue foranother, such as aspartic acid for glutamic acid, or onepositively-charged residue for another, such as lysine for arginine, canalso be expected to produce a biologically equivalent product. See,e.g., FIG. 1.8 on page 10 of Lewin (1997), showing the nature of theside chains of the “standard” 20 amino acids encoded by the geneticcode. (Note also a typographical error in that published figure, namelythat the abbreviation for glutamine should be “Gln.”)

REFERENCES

-   1. Gunning, P., Leavitt, J., Muscat, G., Ng, S. Y. &    Kedes, L. (1987) A human beta-actin expression vector system directs    high-level accumulation of antisense transcripts. Proc Natl Acad Sci    USA 84: 4831-4835.-   2. Reaven, G. M. (1995) Pathophysiology of insulin resistance in    human disease. Physiol Rev 75: 473-486.-   3. Horton, J. D., Goldstein, J. L. & Brown, M. S. (2002) SREBPs:    activators of the complete program of cholesterol and fatty acid    synthesis in the liver. J Clin Invest 109: 1125-1131.-   4. Shimomura, I., Matsuda, M., Hammer, R. E., Bashmakov, Y.,    Brown, M. S. & Goldstein, J. L. (2000) Decreased IRS-2 and increased    SREBP-1c lead to mixed insulin resistance and sensitivity in livers    of lipodystrophic and ob/ob mice. Mol Cell 6: 77-86.-   5. Gavrilova, O., Haluzik, M., Matsusue, K., Cutson, J. J., Johnson,    L., Dietz, K. R., Nicol, C., Vinson, C., Gonzalez, F. &    Reitman, M. L. (2003) Liver PPARgamma contributes to hepatic    steatosis, triglyceride clearance, and regulation of body fat mass.    J Biol. Chem.-   6. Matsusue, K., Haluzik, M., Lambert, G., Yim, S. H., Gavrilova,    0., Ward, J. M., Brewer, B., Jr., Reitman, M. L. &    Gonzalez, F. J. (2003) Liver-specific disruption of PPARgamma in    leptin-deficient mice improves fatty liver but aggravates diabetic    phenotypes. J Clin Invest 111: 737-747.-   7. Yahagi, N., Shimano, H., Hasty, A. H., Matsuzaka, T., Ide, T.,    Yoshikawa, T., Amemiya-Kudo, M., Tomita, S., Okazaki, H. et    al. (2002) Absence of sterol regulatory element-binding protein-1    (SREBP-1) ameliorates fatty livers but not obesity or insulin    resistance in Lep(ob)/Lep(ob) mice. J Biol Chem 277: 19353-19357.-   8. Araki, E., Lipes, M. A., Patti, M. E., Bruning, J. C., Haag, B.,    3rd, Johnson, R. S. & Kahn, C. R. (1994) Alternative pathway of    insulin signalling in mice with targeted disruption of the IRS-1    gene. Nature 372: 186-190.-   9. Withers, D. J., Gutierrez, J. S., Towery, H., Burks, D. J.,    Ren, J. M., Previs, S., Zhang, Y., Bernal, D., Pons, S. et    al. (1998) Disruption of IRS-2 causes type 2 diabetes in mice.    Nature 391: 900-904.-   10. Kido, Y., Burks, D. J., Withers, D., Bruning, J. C., Kahn, C.    R., White, M. F. & Accili, D. (2000) Tissue-specific insulin    resistance in mice with mutations in the insulin receptor, IRS-1,    and IRS-2. J Clin Invest 105: 199-205.-   11. Previs, S. F., Withers, D. J., Ren, J. M., White, M. F. &    Shulman, G. I. (2000) Contrasting effects of IRS-1 versus IRS-2 gene    disruption on carbohydrate and lipid metabolism in vivo. J Biol Chem    275: 38990-38994.-   12. White, M. F. (2002) IRS proteins and the common path to    diabetes. Am J Physiol Endocrinol Metab 283: E413-422.-   13. Ravussin, E. & Smith, S. R. (2002) Increased fat intake,    impaired fat oxidation, and failure of fat cell proliferation result    in ectopic fat storage, insulin resistance, and type 2 diabetes    mellitus. Ann N Y Acad Sci 967: 363-378.-   14. Lowell, B. B. & Shulman, G. I. (2005) Mitochondrial dysfunction    and type 2 diabetes. Science 307: 384-387.-   15. Sutton, G. M., Trevaskis, J. L., Hulver, M. W., McMillan, R. P.,    Markward, N. J., Babin, M. J., Meyer, E. A. & Butler, A. A. (2006)    Diet-genotype interactions in the development of the obese,    insulin-resistant phenotype of C57BL/6J mice lacking melanocortin-3    or -4 receptors. Endocrinology 147: 2183-2196.-   16. Elmquist, J. K., Coppari, R., Balthasar, N., Ichinose, M. &    Lowell, B. B. (2005) Identifying hypothalamic pathways controlling    food intake, body weight, and glucose homeostasis. J Comp Neurol    493: 63-71.-   17. Asilmaz, E., Cohen, P., Miyazaki, M., Dobrzyn, P., Ueki, K.,    Fayzikhodjaeva, G., Soukas, A. A., Kahn, C. R., Ntambi, J. M. et    al. (2004) Site and mechanism of leptin action in a rodent form of    congenital lipodystrophy. J Clin Invest 113: 414-424.-   18. Morton, G. J., Blevins, J. E., Williams, D. L., Niswender, K.    D., Gelling, R. W., Rhodes, C. J., Baskin, D. G. &    Schwartz, M. W. (2005) Leptin action in the forebrain regulates the    hindbrain response to satiety signals. J Clin Invest 115: 703-710.-   19. Coppari, R., Ichinose, M., Lee, C. E., Pullen, A. E., Kenny, C.    D., McGovern, R. A., Tang, V., Liu, S. M., Ludwig, T. et al. (2005)    The hypothalamic arcuate nucleus: A key site for mediating leptin's    effects on glucose homeostasis and locomotor activity. Cell    Metabolism 1: 63-72.-   20. Minokoshi, Y., Kim, Y.-B., Peroni, O. D., Fryer, L. G. D.,    Muller, C., Carling, D. & Kahn, B. B. (2002) Leptin stimulates    fatty-acid oxidation by activating AMP-activated protein kinase.    Nature 415: 339-343.-   21. Roden, M., Price, T. B., Perseghin, G., Petersen, K. F.,    Rothman, D. L., Cline, G. W. & Shulman, G. I. (1996) Mechanism of    free fatty acid-induced insulin resistance in humans. J Clin Invest    97: 2859-2865.-   22. Dresner, A., Laurent, D., Marcucci, M., Griffin, M. E., Dufour,    S., Cline, G. W., Slezak, L. A., Andersen, D. K., Hundal, R. S. et    al. (1999) Effects of free fatty acids on glucose transport and    IRS-1-associated phosphatidylinositol 3-kinase activity. J Clin    Invest 103: 253-259.-   23. Kim, Y. B., Shulman, G. I. & Kahn, B. B. (2002) Fatty acid    infusion selectively impairs insulin action on Akt1 and protein    kinase C lambda/zeta but not on glycogen synthase kinase-3. Biol    Chem 277: 32915-32922.-   24. Roden, M., Krssak, M., Stingl, H., Gruber, S., Hofer, A.,    Furnsinn, C., Moser, E. & Waldhausl, W. (1999) Rapid impairment of    skeletal muscle glucose transport/phosphorylation by free fatty    acids in humans. Diabetes 48: 358-364.-   25. Yu, C., Chen, Y., Cline, G. W., Zhang, D., Zong, H., Wang, Y.,    Bergeron, R., Kim, J. K., Cushman, S. W. et al. (2002) Mechanism by    which fatty acids inhibit insulin activation of insulin receptor    substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase    activity in muscle. J Biol Chem 277: 50230-50236.-   26. Rajala, M. W. & Scherer, P. E. (2003) Minireview: The    adipocyte—at the crossroads of energy homeostasis, inflammation, and    atherosclerosis. Endocrinology 144: 3765-3773.-   27. Kadowaki, T. & Yamauchi, T. (2005) Adiponectin and adiponectin    receptors. Endocr Rev 26: 439-451.-   28. Flier, J. S. (2004) Obesity wars. Molecular progress confronts    an expanding epidemic. Cell 116: 337-350.-   29. Nishizawa, H., Matsuda, M., Yamada, Y., Kawai, K., Suzuki, E.,    Makishima, M., Kitamura,-   T. & Shimomura, I. (2004) Musclin, a novel skeletal muscle-derived    secretory factor. J Biol Chem 279: 19391-19395.-   30. Oike, Y., Akao, M., Yasunaga, K., Yamauchi, T., Morisada, T.,    Ito, Y., Urano, T., Kimura, Y., Kubota, Y. et al. (2005)    Angiopoietin-related growth factor antagonizes obesity and insulin    resistance. Nat Med 11: 400-408.-   31. Sutton, G. M., Trevaskis, J. L., Hulver, M. W., MacMillan, R.    P., Markward, N. J., Meyer, E. A., Babin, M. J. &    Butler, A. A. (2006) Diet-Genotype Interactions in the Development    of the Obese, Insulin Resistant Phenotype of C57BL/6J mice lacking    Melanocortin-3 or -4 Receptors. Endocrinology 147: 2183-2196.-   32. Albarado, D. C., McClaine, J., Stephens, J. M., Mynatt, R. L.,    Ye, J., Bannon, A. W., Richards, W. G. & Butler, A. A. (2004)    Impaired coordination of nutrient intake and substrate oxidation in    melanocortin-4 receptor knockout mice. Endocrinology 145: 243-252.-   33. Browning, J. D. & Horton, J. D. (2004) Molecular mediators of    hepatic steatosis and liver injury. J Clin Invest 114: 147-152.-   34. Sparks, L. M., Xie, H., Koza, R. A., Mynatt, R., Hulver, M. W.,    Bray, G. A. & Smith, S. R. (2005) A high-fat diet coordinately    downregulates genes required for mitochondrial oxidative    phosphorylation in skeletal muscle. Diabetes 54: 1926-1933.-   35. Teran-Garcia, M., Rankinen, T., Koza, R. A., Rao, D. C. &    Bouchard, C. (2005) Endurance training-induced changes in insulin    sensitivity and gene expression. Am J Physiol Endocrinol Metab 288:    E1168-1178.-   36. Koza, R. A., Nikonova, L., Hogan, J., Rim, J. S., Mendoza, T.,    Faulk, C., Skaf, J. & Kozak, L. P. (2006) Changes in gene expression    foreshadow diet-induced obesity in genetically identical mice. PLoS    Genet. 2: e81.-   37. Clark, H. F., Gurney, A. L., Abaya, E., Baker, K., Baldwin, D.,    Brush, J., Chen, J., Chow, B., Chui, C. et al. (2003) The secreted    protein discovery initiative (SPDI), a large-scale effort to    identify novel human secreted and transmembrane proteins: a    bioinformatics assessment. Genome Res 13: 2265-2270.-   38. Kay, M. A., Glorioso, J. C. & Naldini, L. (2001) Viral vectors    for gene therapy: the art of turning infectious agents into vehicles    of therapeutics. Nat Med 7: 33-40.-   39. Chen, G., Koyama, K., Yuan, X., Lee, Y., Zhou, Y. T., O'Doherty,    R., Newgard, C. B. & Unger, R. H. (1996) Disappearance of body fat    in normal rats induced by adenovirus-mediated leptin gene therapy.    Proc Natl Acad Sci USA 93: 14795-14799.-   40. Satoh, H., Nguyen, M. T., Trujillo, M., Imamura, T., Usui, I.,    Scherer, P. E. & Olefsky, J. M. (2005) Adenovirus-mediated    adiponectin expression augments skeletal muscle insulin sensitivity    in male Wistar rats. Diabetes 54: 1304-1313.-   41. Satoh, H., Nguyen, M. T., Miles, P. D., Imamura, T., Usui, I. &    Olefsky, J. M. (2004) Adenovirus-mediated chronic    “hyper-resistinemia” leads to in vivo insulin resistance in normal    rats. J Clin Invest 114: 224-231.-   42. Xu, A., Wang, Y., Keshaw, H., Xu, L. Y., Lam, K. S. &    Cooper, G. J. (2003) The fat-derived hormone adiponectin alleviates    alcoholic and nonalcoholic fatty liver diseases in mice. J Clin    Invest 112: 91-100.-   43. Yamauchi, T., Kamon, J., Minokoshi, Y., Ito, Y., Waki, H.,    Uchida, S., Yamashita, S., Noda, M., Kita, S. et al. (2002)    Adiponectin stimulates glucose utilization and fatty-acid oxidation    by activating AMP-activated protein kinase. Nat Med 8: 1288-1295.-   44. George, S., Rochford, J. J., Wolfrum, C., Gray, S. L., Schinner,    S., Wilson, J. C., Soos, M. A., Murgatroyd, P. R., Williams, R. M.    et al. (2004) A family with severe insulin resistance and diabetes    due to a mutation in AKT2. Science 304: 1325-1328.-   45. Bae, S. S., Cho, H., Mu, J. & Birnbaum, M. J. (2003)    Isoform-specific regulation of insulin-dependent glucose uptake by    Akt/protein kinase B. J Biol Chem 278: 49530-49536.-   46. Trevaskis, J. L. & Butler, A. A. (2005) Double leptin (Lepob)    and melanocortin-4 receptor (Mc4r) gene mutations have an additive    effect on fat mass, and are associated with reduced effects of    leptin on weight loss and food intake. Endocrinology 146: 4257-4265.-   47. Clegg, D. J., Brown, L. M., Woods, S. C. & Benoit, S. C. (2006)    Gonadal hormones determine sensitivity to central leptin and    insulin. Diabetes 55: 978-987.-   48. Clegg, D. J., Riedy, C. A., Smith, K. A., Benoit, S. C. &    Woods, S. C. (2003) Differential sensitivity to central leptin and    insulin in male and female rats. Diabetes 52: 682-687.-   49. Butler, A. A., Blakesley, V. A., Koval, A., deJong, R.,    Groffen, J. & LeRoith, D. (1997) In vivo regulation of CrkII and    CrkL proto-oncogenes in the uterus by insulin-like growth factor-I.    Differential effects on tyrosine phosphorylation and association    with paxillin. J Biol Chem 272: 27660-27664.-   50. Butler, A. A. (2006) The melanocortin system and energy balance.    Peptides 27: 301-309.-   51. Aizawa-Abe, M., Ogawa, Y., Masuzaki, H., Ebihara, K., Satoh, N.,    Iwai, H., Matsuoka, N., Hayashi, T., Hosoda, K. et al. (2000)    Pathophysiological role of leptin in obesity-related hypertension. J    Clin Invest 105: 1243-1252.-   52. Ogawa, Y., Masuzaki, H., Hosoda, K., Aizawa-Abe, M., Suga, J.,    Suda, M., Ebihara, K., Iwai, H., Matsuoka, N. et al. (1999)    Increased glucose metabolism and insulin sensitivity in transgenic    skinny mice overexpressing leptin. Diabetes 48: 1822-1829.-   53. Combs, T. P., Pajvani, U. B., Berg, A. H., Lin, Y., Jelicks, L.    A., Laplante, M., Nawrocki, A. R., Rajala, M. W., Parlow, A. F. et    al. (2004) A transgenic mouse with a deletion in the collagenous    domain of adiponectin displays elevated circulating adiponectin and    improved insulin sensitivity. Endocrinology 145: 367-383.-   54. Yamauchi, T., Kamon, J., Waki, H., Imai, Y., Shimozawa, N.,    Hioki, K., Uchida, S., Ito, Y., Takakuwa, K. et al. (2003) Globular    adiponectin protected ob/ob mice from diabetes and ApoE-deficient    mice from atherosclerosis. J Biol Chem 278: 2461-2468.-   55. Collins, S., Martin, T. L., Surwit, R. S. & Robidoux, J. (2004)    Genetic vulnerability to diet-induced obesity in the C57BL/6J mouse:    physiological and molecular characteristics. Physiol Behav 81:    243-248.-   56. Bates, S. H., Kulkarni, R. N., Seifert, M. & Myers, M. G.,    Jr. (2005) Roles for leptin receptor/STAT3-dependent and    -independent signals in the regulation of glucose homeostasis. Cell    Metab 1: 169-178.-   57. Shimomura, I., Bashmakov, Y. & Horton, J. D. (1999) Increased    levels of nuclear SREBP-1c associated with fatty livers in two mouse    models of diabetes mellitus. J Biol Chem 274: 30028-30032.-   58. Masuzaki, H., Ogawa, Y., Aizawa-Abe, M., Hosoda, K., Suga, J.,    Ebihara, K., Satoh, N., Iwai, H., Inoue, G. et al. (1999) Glucose    metabolism and insulin sensitivity in transgenic mice overexpressing    leptin with lethal yellow agouti mutation: usefulness of leptin for    the treatment of obesity-associated diabetes. Diabetes 48:    1615-1622.-   59. Heisler, L. K., Jobst, E. E., Sutton, G. M., Zhou, L., Borok,    E., Thornton-Jones, Z., Liu, H. Y., Zigman, J. M., Balthasar, N. et    al. (2006) Serotonin Reciprocally Regulates Melanocortin Neurons to    Modulate Food Intake. NeuronJul 20; 51(2):239-249.-   60. Butler, A. A., Kesterson, R. A., Khong, K., Cullen, M. J.,    Pelleymounter, M. A., Dekoning, J., Baetscher, M. &    Cone, R. D. (2000) A unique metabolic syndrome causes obesity in the    melanocortin-3 receptor-deficient mouse. Endocrinology 141:    3518-3521.

The complete disclosures of all references cited in this specificationare hereby incorporated by reference. In the event of an otherwiseirreconcilable conflict, however, the present specification shallcontrol.

What is claimed:
 1. A Enho1 peptide, wherein the sequence of the Enho1peptide is a sequence selected from the group consisting of SEQ ID NO:10 and SEQ ID NO:11.
 2. The peptide of claim 1, wherein the sequence isSEQ ID NO:
 10. 3. The peptide of claim 1, wherein the sequence is SEQ IDNO:
 11. 4. The peptide of claim 1, wherein the peptide is a mammalianpeptide.
 5. The peptide of claim 1, wherein the peptide is a humanpeptide.
 6. The peptide of claim 1, wherein the peptide is a murinepeptide.
 7. The peptide of claim 1, wherein the peptide is a syntheticpeptide.
 8. An antibody, and fragments, derivatives, homologs andanalogs thereof, which immunospecifically binds to the peptide ofclaim
 1. 9. An antibody of claim 8 labeled with a detectable label. 10.An antibody or fragment of claim 8, wherein the antibody is a polyclonalantibody.
 11. An antibody or fragment of claim 8, wherein the antibodyis a monoclonal antibody.
 12. A pharmaceutical composition comprising anEnho1 peptide of claim 1, and a pharmaceutical acceptable carrier. 13.The pharmaceutical composition of claim 8, additionally comprising acompound selected from the group consisting of leptin and adiponectin.